- Methods of RNA preparation affect mRNA abundance quantification of
reference genes in pig maturing oocytes
- Authors: Y-K Wang; X Li, Z-Q Song, C-X Yang
Abstract: To ensure accurate normalization and quantification of target RNA transcripts using reverse transcription quantitative polymerase chain reaction (RT-qPCR), most studies focus on the identification of stably expressed gene(s) as internal reference. However, RNA preparation methods could also be an important factor, especially for test samples of limited quantity (e.g. oocytes). In this study, we aimed to select appropriate reference gene(s), and evaluate the effect of RNA preparation methods on gene expression quantification in porcine oocytes and cumulus cells during in vitro maturation. Expression profiles of seven genes (GAPDH, 18S, YWHAG, BACT, RPL4, HPRT1 and PPIA) were examined, on RNA samples extracted from cumulus cells (RNeasy Kit) and oocytes (RNeasy Kit and Lysis Kit) during in vitro maturation, respectively. Interestingly, different RNA preparation methods were found to potentially affect the quantification of reference gene expression in pig oocytes cultured in vitro. After geNorm analyses, the most suitable genes for normalization were identified, GAPDH/18S for cumulus cells and YWHAG/BACT for oocytes, respectively. Thus, our results provide useful data and information on the selection of better reference genes and RNA preparation method for related functional studies.
- Endometrial gene expression in primiparous dairy cows at the end of the
voluntary waiting period is affected by nutrition: Total mixed ration vs
increasing levels of herbage allowance
- Authors: AL Astessiano; M Carriquiry, DA Mattiauda, ML Adrien, P Chilibroste, A Meikle
Abstract: The study postulated that differential nutritional management during the early lactation period would be reflected in endometrial expression of genes related to embryo growth at the end of the voluntary waiting period. Thus, the effect of the combined use of total mixed ration (TMR) and grazing under different herbage allowances during the first 75 days post-partum (DPP) on endometrial gene expression was evaluated in primiparous dairy cows. Cows were blocked by body weight, age and body condition score and randomly assigned to three grazing treatments: high (HA, 30 kg DM per cow per day), medium (MA, 15 kg DM per cow per day) and low (LA, 7.5 kg DM per cow per day) herbage allowance (mixed pasture, 2,600 kg DM per ha) plus 8 kg DM of supplement or TMR (55% forage, 45% concentrate) fed ad libitum (TMR) from calving to 75 DPP. At 57 DPP, cows were synchronized for oestrus (day 0, 68 DPP) and at day 7, endometrial biopsies were obtained. The nutritional treatment did not affect insulin, IGF-1 and leptin concentrations on days 0, 4 or 7. Expression of IGF1, IGFBP3, IGFBP4, ADIPOR1 and ADIPOR2 mRNA was significantly affected by the nutritional treatment. Endometrial IGF1 and IGFBP4 mRNA were twofold greater in TMR and HA than MA and LA cows. Expression of IGFBP3 and ADIPOR1 mRNAs was greater in TMR and HA than MA cows, but did not differ from LA cows. All groups had greater expression of ADIPOR2 mRNA than MA cows. This study provided solid evidence of the importance of nutritional management during early lactation on uterine environment at the end of the voluntary waiting period. The greater expression of genes related to embryo growth and uterine function (IGF system, progesterone and adiponectin receptors) in cows fed diets maximizing energy intake suggests a favourable environment for embryonic growth, which may explain the improved reproductive performance of cows in good energy balance.
- Validation of the BioPRYN enzyme-linked immunosorbent assay for detection
of pregnancy-specific protein-B (PSPB) and diagnosis of pregnancy in
American bison (Bison bison)
- Authors: DM Love; MT Mefford, JC Ramer
Abstract: This study assessed the accuracy of the commercial BioPRYN® ELISA for the detection of pregnancy-specific protein-B (PSPB) using a single blood sample to determine pregnancy status in American bison (Bison bison). A total of 49 bison cows were used in the study, and sampled at two time-points during the gestation period, fall and spring, correlating with early- to mid-term gestation (average 62.9 days post-mating) and mid- to late-term gestation (average 229.2 days post-mating), respectively. Sensitivity of the test during early- to mid-term gestation sampling period (fall) was 87.1%, while specificity was 100%; sensitivity of the test during late-term gestation sampling period (spring) was 96.3%, while specificity remained at 100%. In total, the test showed a total sensitivity of 91.4%, specificity of 100% and total accuracy of 93.8%, similar to domestic cattle. Use of the single-sample BioPRYN® ELISA in American Bison for pregnancy diagnosis is economical and practical, minimizing animal handling time, frequency and subsequent stress while providing accurate results for pregnancy diagnosis at 62 days post-mating. This method should be considered over more traditional pregnancy diagnosis methods for use in managed bison herds.
- Participation of phosphofructokinase, malate dehydrogenase and isocitrate
dehydrogenase in capacitation and acrosome reaction of boar spermatozoa
- Authors: E Breininger; D Dubois, VE Pereyra, PC Rodriguez, MM Satorre, PD Cetica
Abstract: The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.
- Effect of rapamycin treatment during post-activation and/or in vitro
culture on embryonic development after parthenogenesis and in vitro
fertilization in pigs
- Authors: F Elahi; H Lee, J Lee, ST Lee, CK Park, S-H Hyun, E Lee
Abstract: This study investigated the effects of early induction of autophagy on embryonic development in pigs. For this, oocytes or embryos were treated with an autophagy inducer, rapamycin (RP), during post-activation (Pa), in vitro fertilization (IVF) and/or in vitro culture (IVC). When parthenogenesis (PA) embryos were untreated (control) or treated with various concentrations of RP for 4 hr during Pa, 100 nm RP showed a higher blastocyst formation (48.8 ± 2.7%) than the control (34.6 ± 3.0%). When PA embryos were treated during the first 24 hr of IVC, blastocyst formation was increased (p
- Colostrum yield and litter performance in multiparous sows subjected to
- Authors: MA Otto; AP Machado, LP Moreira, ML Bernardi, ML Coutinho, IS Vaz, I Wentz, FP Bortolozzo
Abstract: The consumption of colostrum at a low level can compromise the survival and growth of piglets. Therefore, this study aimed to evaluate the effect of farrowing induction on colostrum yield, IgG concentration and the survival and performance of piglets until the weaning. Sows of parity 3 to 7 were assigned into two groups: Control (n = 48), sows with spontaneous farrowing; and induction (n = 48), sows induced to farrow on day 114 of gestation with a PGF2 analogue. Colostrum and blood samples were collected from the sows, at farrowing and 24 hr later. Blood samples from the piglets were collected at 24 hr after birth. The performance of the piglets was evaluated in a subsample of 28 litters from each group. All piglets were weighed at 7, 14 and 20 days of age. The farrowing length, the number of piglets born alive, stillborn piglets, weight at birth, litter weight at birth and colostrum yield were not significantly affected (p > .05) by farrowing induction. There was no difference between the groups (p > .05) in the percentage of sows with obstetric interventions. Serum IgG concentration, in both sows and piglets, and colostrum IgG concentration were similar between the groups (p > .05). Furthermore, survival rate, piglet weight and litter weight at 7, 14 and 20 days of age were also similar between the groups (p > .05). Therefore, it can be concluded that the farrowing induction performed on day 114 of gestation does not affect the colostrum yield, the IgG concentration in colostrum and serum of piglets, and the litter performance until the weaning.
- Luteal activity and effect of dietary energy restriction on follicular
development in lactating cows
- Authors: A Comin; T Peric, M Montillo, A Cappa, V Marchi, MC Veronesi, A Prandi
Abstract: The aim of this research has been to evaluate the presence of anomalies in the ovarian cycle activity during postpartum and to verify whether 72-hr dietary fasting during the dominance phase, the phase before ovulation, might modify the ovarian follicle population. The presence of anomalies in ovarian cycle activity has been evaluated in 30 Italian Friesian cows starting from 20 days postpartum until 211 days of lactation. Long oestrus and brief dioestrus or scarce luteal activity have been the main anomalies found through measuring progesterone concentrations in the whey. Until 100 days of lactation, the BCS values of the problematic animals have been significantly lower than those in animals with normal ovarian activity. After 100 days of lactation, the ovarian anomalies continued to appear despite the fact that all the animals have reached comparable BCS values. Starting from the results of this trial, the effect of 72-hr dietary fasting on dominant follicles has been studied in six cows. Ultrasonography revealed that the diameter of the follicles at 71 days postpartum has been significantly lower than at 181 days. A 72-hr dietary restriction at 101 and 211 days postpartum did not affect the size of the dominant follicle. However, at 101 days postpartum, half of the animals presented follicular cysts. The effect of fasting differed if the animal has been in early postpartum or 211 days of lactation. Further researches are necessary to understand how different metabolic conditions can modify the follicular population but on the other hand the study shows the utility for farmers and field veterinarians of monitoring the resumption of the ovarian cycle postpartum through the whey progesterone concentrations.
- Expression of kit ligand and insulin-like growth factor binding protein 3
during in vivo or in vitro development of ovarian follicles in sheep
- Authors: D Srividya; V Praveen Chakravarthi, SSR Kona, AVN Siva Kumar, KV Brahmaiah, VH Rao
Abstract: Expression of Kit ligand (KL) and insulin-like growth factor binding protein (IGFBP3) genes was studied at different in vivo and corresponding in vitro stages of development of the ovarian follicles in sheep. The expression of both KL and IGFBP3 was significantly higher in the primordial follicles relative to any other stage of development. Compared to the other stages, the KL expression in the cumulus cells from in vivo grown large antral follicles and that of IGFBP3 in COCs’ isolated from large antral follicles matured in vitro for 24 hr were significantly higher. In the oocytes from in vivo grown ovarian follicles, the expression of KL was the same at all the stages of development. Insulin-like growth factor binding protein 3 expression was also the same in the oocytes at all the stages of the development except for a significantly lower expression in those from antral follicles. The expression of KL in the cumulus cells decreased significantly in the in vitro grown early antral follicles but did not change further as the development progressed. The expression of IGFBP3 in the cumulus cells from in vitro grown ovarian follicles appeared to increase as the development progressed although the increase was not significant between any two consecutive stages of development. In the oocytes in in vitro grown ovarian follicles, the expression levels of KL and IGFBP3 genes did not change with development. It is concluded that (i) KL and IGFBP3 genes follow specific patterns of expression during ovarian folliculogenesis and (ii) in vitro culture of preantral follicles compromises the development potential through alterations in the stage-specific patterns of expression of these and other developmentally important genes.
- Aquaporin 9 is expressed in the epididymis of immature and mature pigs
- Authors: BC Schimming; CAE Baumam, PFF Pinheiro, R Matteis, RF Domeniconi
Abstract: Aquaporins (AQPs) are channel proteins that facilitate the transepithelial and bidirectional movement of water. AQP9 is an aquaporin that is expressed in the mammalian epididymis. This water transport contributes to epididymal sperm concentration. This study aimed to examine the morphology of epididymal epithelium in piglets and boars, as well as the expression and immunolocalization of AQP9. The piglets presented an epididymal epithelium in differentiation with principal, basal and apical cells. The cellular population of the epididymal epithelium in boars consisted of principal, basal, apical, clear and narrow cells. The migratory cells known as halo cells were observed in the epididymis of both piglets and boars. AQP9 expression presented differences between piglets and boars. Moderate intensity of AQP9 immunoreaction was observed in the apical border of the epididymal epithelium of the caput and cauda regions in the piglet epididymis. A moderate-to-intense reaction for AQP9 was observed in the nuclei of epithelial cells of the three epididymal regions in the boar epididymis. The region of the cauda epididymis showed reactivity for AQP9 also in the apical border of the epithelium. It is believed that the AQP9 is already functional in piglets at only 1 week of age and is more active, playing a pivotal role in the caput and cauda regions of the epididymis. Moreover, the intense AQP9 expression in the apical border of epithelial cells in the cauda region of the boar epididymis suggests a higher performance of AQP9 in this region, where sperm complete their maturation process, stored and concentrated.
- Disorder of sex development in a cat with chromosome mosaicism
- Authors: I Szczerbal; M Stachowiak, J Nowacka-Woszuk, S Dzimira, K Szczepanska, M Switonski
Abstract: An 18-month-old European shorthair cat was subjected to genetic studies due to ambiguous external genitalia (underdeveloped both penis and scrotum). Further anatomic and histopathological studies revealed the presence of abdominal, atrophic testes and uterus. Cytogenetic analysis showed two cell lines, one with X monosomy—37,X [90% of the analysed metaphase spreads], and other line had 38 chromosomes with normal X chromosome and abnormally small Y-derived chromosome—38,X,der(Y) [10%]. Further fluorescence in situ hybridization study with telomeric probe revealed a ring structure of the der(Y). Eight Y chromosome-specific genes, SRY, TETY1, TETY2, CUL4BY, CYORF15, HSFY, FLJ36031Y and ZFY, were detected. We conclude that the described abnormality of the reproductive system, leading to sterility, was caused by a very rare type of chromosomal mosaicism—37,X/38,X,r(Y).
- Supplementation with cumulus cell masses improves the in vitro meiotic
competence of porcine cumulus–oocytes complexes derived from small
- Authors: R Matsunaga; H Funahashi
Abstract: The present study was conducted to examine the supplemented effect of cumulus cell masses (CCMs) derived from middle follicle (MF; 3–6 mm diameter) on the morphology and the meiotic or developmental competence of oocytes from small follicles (SF; 1–2 mm diameter). The number of cumulus cells surrounding oocytes just after collection was also lower in cumulus–oocyte complexes (COCs) from SF than MF. The ooplasmic diameter of oocytes was significantly smaller in SF-derived oocytes than MF-derived ones before and after in vitro maturation (IVM), whereas the diameter significantly increased during the culture. Co-culture of SF-derived COCs with MF-derived CCMs during IVM significantly improved the meiotic competence of the oocytes to the metaphase-II stage. Furthermore, the ooplasmic diameter of SF-derived COCs during IVM was increased to the similar size of MF-derived those in the presence of MF-derived CCMs. The abilities of oocytes to be penetrated, to form male pronuclear formation and to cleave or develop to the blastocyst stage were not affected by the co-culture with CCMs. Electrophoretic analysis of CCM secretions clearly showed the presence of more protein(s) approximately 27.6 kDa in the conditioned medium when supplemented with MF-derived CCMs. In conclusion, we demonstrate that supplementation with MF-derived CCMs improves the ooplasmic diameter and meiotic competence of SF-derived oocytes.
- Genetic differences in oestrous signs and oestrogen metabolism-related
genes between Chinese Mi and European Landrace-Large White pigs
- Authors: Q Chu; T Liang, B Zhou
Abstract: Oestrous signs affect timely mating and reproductive efficiency in swine breeding herds. To study the genetic difference of oestrous signs between Chinese and European pigs, 100 Landrace-Large White (LLW) cross gilts and 50 Chinese Mi gilts were assessed for oestrous signs and the concentrations of serum estradiol-17β and progesterone were determined. The genotype of 39 single nucleotide polymorphisms (SNPs) in 11 oestrogen metabolism and function-related genes was determined by Sequenom iPLEX platform. Compared with LLW gilts, Mi gilts had longer time of standing reflex (p
- Role of prostatic fluid in cooled canine epididymal sperm
- Authors: T Hori; T Masuda, M Kobayashi, E Kawakami
Abstract: In this study, sperms collected from the right and left cauda epididymis were grouped into having canine prostatic fluid (PF) sensitization or not diluted with egg yolk Tris–fructose citrate extender, and stored at 4°C. The necessity of canine PF in cooled preservation was determined by elucidating the sperm quality after the storage. As a result, while there was no difference among all groups up to 48 hr of storage, after storage for 96 hr and more, a significantly lower sperm motility was observed in the group without being sensitized to PF than the groups with being sensitized to PF (p
- GDF9 affects the development and tight junction functions of immature
bovine Sertoli cells
- Authors: K Tang; L Wang, Y Jin, W Yang, L Yang
Abstract: Transforming growth factor-β (TGFβ) superfamily are critical regulators of germ cell development that act as extracellular ligands of the signal transduction pathways regulating proliferation, apoptosis and other aspects of cell behaviour. As a member of the TGF-β superfamily, growth differentiation factor 9 (GDF9) plays a critical role in ovarian follicular development and the ovulation rate in females; however, its role in the testis has not been well elucidated. The aim of this study was to investigate the expression of GDF9 and its receptor genes BMPRII and ALK5 in prepuberal bovine Sertoli cells (SCs). In addition, we assessed the effects of GDF9 on immature SCs apoptosis, the cell cycle and tight junction functions. We found that GDF9 and its receptor genes BMPRII and ALK5 were expressed in immature SCs. Exogenous GDF9 significantly promoted SCs proliferation and inhibited the apoptosis of SCs by significantly upregulating Cyclin E (cell cycle) and bcl-2 (anti-apoptosis) mRNA expression and downregulating caspase-3 (pro-apoptosis) mRNA expression. Meanwhile, exogenous GDF9 significantly decreased the value of transepithelial electrical resistance by significantly downregulating claudin-11 mRNA expression and influencing the distribution of occludin. In conclusion, this study reveals that GDF9 is a key regulator of bovine SCs through the modulation of the cell cycle, apoptosis and tight junction functions.
- Ultrasound during mid-gestation: Agreement with physical foetal and
placental measurements and use in predicting gestational age in sheep
- Authors: AK Jones; RE Gately, KK McFadden, ML Hoffman, SM Pillai, SA Zinn, KE Govoni, SA Reed
Abstract: To determine the effects of poor maternal nutrition and litter size on foetal growth during mid-gestation, pregnant ewes (n = 82) were fed 100%, 60% or 140% of NRC TDN beginning at day 30.2 ± 0.2 of gestation. Transabdominal ultrasound was performed weekly between day 46.0 ± 0.4 and 86.0 ± 0.7 to monitor foetal heart width (HW), umbilical diameter (UMB), rib width (RW) and placentome outer (OD) and inner diameter (ID). Data were analysed with repeated-measures using the mixed procedure for effects of maternal diet, litter size and gestation, and equations predictive of gestational age were generated using the regression procedure. To determine the agreement of ultrasound measurement and actual size, ewes (n = 20–21) were euthanized at day 45 or 90 to obtain corresponding postmortem measurements for Bland–Altman analysis. The HW, UMB and placentome OD and ID increased with gestation (p
- Bilateral oblique facial clefts, rudimentary eyes and hydrocephalus in an
aborted equine foetus
- Authors: JS Agerholm; HG Pedersen, FJ McEvoy, S Heegaard
Abstract: Knowledge of congenital malformations and their causes in horses is generally sparse. Such conditions require more scientific attention to improve their diagnostics and inform prevention strategies. Here, a unique syndrome of bilateral oblique facial clefts (meloschisis), rudimentary eyes and hydrocephalus is reported in an equine foetus spontaneously aborted at gestation day 224. The cause of abortion was considered to be intrauterine death caused by umbilical cord torsions and subsequent compromised blood flow, but the aetiology of the malformation could not be determined. A detailed history, which includes exposure to a range of pharmaceutical compounds during the early stages of pregnancy, is provided and emphasizes the need for accurate recording of treatments in pregnant animals.
- A comparative study of the effects of intramuscular administration of
gonadorelin, mating and intrauterine infusion of either raw seminal plasma
or seminal plasma purified β-NGF on luteal development in llamas
- Authors: M Silva; F Urra, C Ulloa-Leal, MH Ratto
Abstract: The aim of this study was to compare the effect of the intramuscular administration of 50 μg of gonadorelin acetate versus natural mating, intrauterine infusion (i.u.) of a physiological relevant dose of either raw llama seminal plasma (SP) or purified beta-nerve growth factor from seminal origin (spβ-NGF) on ovulation rate and corpus luteum (CL) development and function in llamas. Females with a follicle (≥8 mm) were assigned to groups: (i) i.m. administration of 50 μg of gonadorelin acetate (GnRH; positive control; n = 4); (ii) single mating (mating; n = 6); (iii) i.u. infusion of 4 ml of llama SP (SP; n = 4); or (iv) i.u. infusion of 10 mg of spβ-NGF contained in 4 ml of PBS (phosphate-buffered saline) (spβ-NGF; n = 6). Ovaries were examined by power Doppler ultrasonography at 0, 1, 3, 6, 12 and 24 hr after treatment to determine preovulatory follicle vascularization area (VA), and additionally every 12 hr until Day 2 (Day of treatment = Day 0) to determine ovulation. Afterwards, ovaries were examined every other day until Day 8 to evaluate CL diameter and VA. Blood samples were collected on Days 0, 2, 4, 6 and 8 to determine plasma progesterone (P4) concentration. Ovulation rate did not differ (p = .7) among groups, but treatment affected (p
- Toll-like receptor and related cytokine mRNA expression in bovine corpora
lutea during the oestrous cycle and pregnancy
- Authors: JE Gadsby; AM Tyson Nipper, HA Faircloth, M D'Annibale-Tolhurst, J Chang, PW Farin, IM Sheldon, DH Poole
Abstract: Improving our understanding of the mechanisms controlling the corpus luteum (CL) and its role in regulating the reproductive cycle should lead to improvements in the sustainability of today's global animal industry. The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture steroidogenic, endothelial and immune cells, and it is becoming clear that immune mechanisms play a key role in CL regulation especially in luteolysis. Toll-like receptors (TLR) mediate innate immune mechanisms via the production of pro-inflammatory cytokines, especially within various tissues, although the role of TLR within CL remains unknown. Thus, the objectives of this study were to characterize TLR mRNA expression in the CL during the oestrous cycle and in pregnancy (day 30–50), and to examine the role of TLR signalling in luteal cells. Corpora lutea were collected at various stages of the cycle and pregnancy and analysed for TLR and cytokine mRNA expression. In addition, luteal cells were cultured with the TLR4 ligand (lipopolysaccharide, LPS) for 24 h to evaluate the role of TLR4 in regulating luteal function. Toll-like receptors 1, 2, 4, 6, tumour necrosis factor alpha (TNF), interferon gamma (IFN-G), and interleukin (IL)-12, mRNA expressions were greatest in regressing CL compared with earlier stages (p
- Reproductive performance of postpartum ewes treated with insulin or
progesterone hormones in association with ram effect
- Authors: JC Ferreira-Silva; SRL Basto, F Tenório Filho, MT Moura, ML Silva Filho, MAL Oliveira
Abstract: The reproductive performance of postpartum Santa Inês (SI) and Morada Nova (MN) ewes treated with insulin or progesterone hormones in association with ram effect was evaluated. Ewes from SI (n = 69) and MN (n = 69) breeds were randomly allocated to three groups of each breed (T1—ram effect only; T2—ram effect + insulin; T3—ram effect + progesterone). Progesterone concentrations (ηg/ml; mean ± SD) before and after introduction of rams (n = 6) were 0.51 ± 0.22 and 3.78 ± 0.68 (T1), 0.65 ± 0.21 and 3.77 ± 0.78 (T2) and 0.52 ± 0.21 and 3.84 ± 0.84 (T3) in SI ewes and 0.74 ± 0.19 and 3.71 ± 0.56 (T1), 0.70 ± 0.21 and 3.79 ± 0.75 (T2) and 0.81 ± 0.14 and 3.87 ± 0.80 (T3) in MN ewes, respectively. Thus, lower progesterone concentrations were found before the introduction of rams (p .05), was 1.3 ± 0.51 (T1), 1.5 ± 0.54 (T2) and 1.6 ± 0.51 (T3) in SI ewes and 1.3 ± 0.51 (T1), 1.6 ± 0.51 (T2) and 1.6 ± 0.51 (T3) in MN ewes. In conclusion, the ram effect alone is effective at inducing and synchronizing oestrus in sheep under postpartum anoestrus, irrespective of hormone treatments.
- Recombinant expression of porcine spermadhesin AWN and its phospholipid
interaction: Indication for a novel lipid binding property
- Authors: F Schröter; K Müller, P Müller, E Krause, BC Braun
Abstract: AWN is a porcine (Sus scrofa domestica) seminal plasma protein and has been linked to a variety of processes related to fertilization. To acquire the protein in sufficient amount and purity for functional studies, we established its recombinant expression in E. coli and a three-step purification protocol based on different chromatographies. The test for AWN–phospholipid interaction revealed phosphatidic acid and cardiolipin as potential binding partners. As phosphatidic acid is surmised to play a role in cation-induced membrane destabilization and fusion events, we propose a membrane protective function of the presented binding affinity. Further studies with recombinant AWN will allow new insights into the mechanism of sperm–spermadhesin interaction and might provide new approaches for artificial reproduction techniques.
- Effects of single layer centrifugation (SLC) on bull spermatozoa prior to
freezing on post-thaw semen characteristics
- Authors: T Nongbua; A Johannisson, A Edman, JM Morrell
Abstract: Single layer centrifugation (SLC) has been shown to select the most robust spermatozoa from the ejaculate in several species. Here the effects of SLC prior to freezing on various parameters of frozen-thawed bovine sperm quality are reported. Semen from 8 bulls was layered on top of a species-specific colloid, Bovicoll. After centrifugation for 20 min at 300 g, the resulting sperm pellet was resuspended in OPTIXcell® (IMV Technologies, l′Aigle, France); the SLC-selected sperm samples and uncentrifuged controls were frozen. On thawing, all sperm samples were analysed for membrane integrity, production of reactive oxygen species, mitochondrial membrane potential (MMP) and chromatin integrity. The SLC-treated samples had a higher percentage of live, superoxide-positive spermatozoa than uncentrifuged samples (27.9 ± 5.1% versus 21.7 ± 6.7%; p = .03). They had a higher proportion of spermatozoa with high mitochondrial membrane potential than uncentrifuged samples (55.9 ± 8.2% versus 40.5 ± 15.1%; p = .03) and also a lower proportion of spermatozoa with low mitochondrial membrane potential than non-treated samples (42.0 ± 8.5% versus 55.9 ± 14.4%; p = .04). No significant effects of treatment were found for membrane integrity or chromatin integrity. The effect of bull was significant on the proportions of dead, superoxide-positive spermatozoa and live, hydrogen peroxide-negative spermatozoa, as well as on membrane integrity, but it was not significant for mitochondrial membrane potential or chromatin integrity. These results suggest that SLC selects the most metabolically active bull spermatozoa from the rest of the population in normal ejaculates; the pattern of reactive oxygen species production may be different in SLC-selected spermatozoa compared to unselected samples.
- Effect of selenium and vitamin E addition to the extender on liquid stored
capercaillie (Tetrao urogallus) semen quality
- Authors: AM Kowalczyk; J Klećkowska-Nawrot, ET Łukaszewicz
Abstract: Captive breeding has become an important tool in species conservations programmes, maintaining genetic diversity and restoring wild, endangered populations. In order to improve the reproductive efficiency of captive kept capercaillie, the purpose of the study was to determine the effect of selenium and vitamin E addition to semen extender on sperm characteristic during short-term storage. Ejaculates collected individually from four capercaillie were divided into two parts, diluted threefold with basic EK extender and EK enriched with 1 mg/ml of organic selenium and 8 mg/ml of vitamin E (EK+Se+E) and stored 24 hr at temp. +4°C. Spermatozoa morphology, motility and motility parameter were evaluated in net, diluted and stored semen samples. Significant (p
- Influence of partial or total replacement of glycerol by alternative
cryoprotectants in Ghent freezing extender on post-thaw sperm quality in
- Authors: RA De Oliveira; S Budik, C Aurich
Abstract: Although glycerol is the cryoprotectant most commonly used in stallions, it has also a considerable toxicity for equine sperm. It was the aim of this study to analyse the quality of frozen-thawed stallion semen after complete or partial replacement of glycerol in the freezing extender by alternative cryoprotectants. We hypothesized that partial or total replacement of glycerol by cryoprotectants occurring in cold-resistant frog, insect or plant species results in similar or better semen quality after freezing–thawing. As basic medium, the commercial Ghent basic extender was used and either supplemented with glucose and urea, trehalose and proline, or trehalose and betaine. Based on a series of preliminary experiments, semen was frozen in either commercial Ghent cryopreservation extender (Ghent control), Ghent glucose–urea extender or a Ghent combined extender (glucose–urea, trehalose-betaine and trehalose-proline; volume ratio of 2:1:2) in a computer-controlled rate freezer. After freezing–thawing, semen was analysed for motility, membrane integrity, phosphatidylserine translocation, mitochondrial membrane potential and chromatin condensation. No differences between Ghent control and Ghent glucose–urea extender were seen, while all endpoints except DNA integrity were negatively affected in Ghent combined extender (e.g., progressive motility: Ghent 49.2 ± 3.7, Ghent glucose–urea 46.5 ± 4.6, Ghent combined 24.4 ± 2.8%; p
- ‘Post-mortem examination of the reproductive organs of female wild boars
(Sus scrofa) in Sweden’
- Authors: Anna Malmsten; Gunnar Jansson, Anne-Marie Dalin
Abstract: In recent decades, wild boars (Sus scrofa) have increased in numbers and distribution in Europe. Compared to other wild ungulates of similar body size, wild boars have a high reproductive capacity. To increase the knowledge of wild boar reproduction, the objective of this study was to investigate characteristics of reproductive organs, and to provide information on the occurrence of abnormalities in reproductive organs from free-ranging female wild boars. Between December 2011 and December 2015, reproductive organs from female wild boars (>30 kg body weight), were collected during hunting in four Swedish counties at estates where supplementary feeding was applied. The organs were macroscopically examined and measured. The stage of the reproductive cycle was defined according to the ovarian structures and in relation to uterus characteristics. Observed abnormalities were noted. The results from 569 animals that met the requirements to be included in this study showed significant differences in weight and length of the uterus between the various reproductive stages. Sampling region had significant effect on these differences. Abnormalities in the reproductive organs were present in approximately 10% of the examined animals. The prevalence of abnormalities increased significantly with age and was significantly affected by sampling region.
- Chilled and post-thaw storage of sperm in different goldfish types
- Authors: G Bernáth; I Ittzés, Z Szabó, Á Horváth, S Krejszeff, J Lujić, L Várkonyi, B Urbányi, Z Bokor
Abstract: The effective storage time of sperm after stripping (for 48 hr in 6-hr intervals) and after thawing (for 6 hr in 2-hr intervals) in Black moor, Oranda and Calico goldfish types was investigated. Variations in sperm density were also measured in all lines. The efficiency of a sperm cryopreservation method formerly developed for common carp was recorded in all three goldfish lines. Motility parameters ((pMOT, %), curvilinear velocity (VCL, μm/s) and straightness (STR, %)) of Black moor sperm did not decrease significantly during 48 hr of storage. A significant reduction in the Oranda type compared to the fresh control was observed in pMOT after 42 (23 ± 2%) and VCL after 36 (94 ± 12 μm/s) hours (pMOT 84 ± 5%, VCL 150 ± 11 μm/s). In the Calico type, pMOT decreased significantly already after 18 (42 ± 26%) and VCL after 6 (105 ± 8 μm/s) hours (fresh: pMOT 92 ± 5%, VCL 151 ± 6 μm/s). A high pMOT immediately following thawing was measured in Oranda (46 ± 12%) and Calico (55 ± 15%) types, whereas a reduced pMOT was recorded in Black moor (24 ± 19%). In Calico, pMOT showed a significant reduction after 6 hr (19 ± 11%) in comparison with the initial value, with no changes observed in VCL and STR. None of the parameters changed in the Black moor and Oranda types. Evidence was found that different goldfish lines have different sperm quality and characteristics. Further studies can investigate the possible effects of chilled and post-thaw storage on the fertilizing capacity of sperm in the Black moor, Oranda and Calico goldfish types.
- Effect of steroid hormones on the innate immune response induced by
Staphylococcus aureus in the goat mammary gland
- Authors: K Kuwahara; Y Yoshimura, N Isobe
Abstract: The objective of this study was to compare the dynamics of innate immune components after intramammary infusion of Staphylococcus aureus (SA) under conditions of high oestrogen and high progesterone in goats. In one group (“E-group”), controlled internal drug release (CIDR) devices were inserted intravaginally from days −11 to −4. Prostaglandin F2α was administered immediately after removal of the CIDR device at day −3, and then oestradiol benzoate (E) was injected intramuscularly once a day from days −2 to 3. Heat-inactivated SA was then administered via intramammary infusion to the left udder at day 0, whilst only saline was infused to the right udder as a control. In a second group (“P-group”), CIDR devices were inserted intravaginally from days −3 to 7 and SA was infused at day 0 in the same way as in the E-group. The milk yield and the concentration of innate immune components (somatic cell count (SCC), lactoferrin (LF), S100A7 and goat ß-defensin 1 (GBD-1)) in the milk were measured. Milk yield decreased drastically in both SA and control udders in the E-group, whereas the P-group exhibited increased milk yield in both SA and control udders. SCC increased after SA infusion in both E- and P-groups, although it was higher in the E-group than in the P-group. There was no significant change in LF concentration in the E-group, but a decrease was observed in the P-group. Concentrations of S100A and GBD-1 were significantly increased after SA infusion in the E-group but not in the P-group. These results suggest that E enhances the innate immune response induced by SA in the goat mammary gland. This effect may be due to the reduction in milk yield and upregulation of innate immune components.
- Polymorphism in 5′ untranslated region of heat-shock protein 70 gene as
marker of post-partum anoestrus in Murrah buffaloes
- Authors: R Kumar; M Ghosh, N Kumar, AK Balhara, M Gupta, RK Sharma, I Singh
Abstract: The enormous production potential of buffaloes has never been accomplished due to various reproductive insufficiencies. Among them, post-partum anoestrus, a multifactorial disorder, is predominant but any genetic association is yet to be established. This study focused to identify novel polymorphisms in heat-shock protein 70 (HSP70) gene and its possible association with post-partum anoestrus in Murrah buffaloes. A 579-bp fragment from 5′ untranslated region of HSP70 gene was amplified by polymerase chain reaction from blood genomic DNA of 614 animals maintained under similar management conditions. In phase-I experiment, custom sequencing and restriction enzyme (RE) digestion of the amplified fragment were performed in 40 buffaloes with similar post-partum oestrous conditions over previous consecutive three or more gestations—20 animals each showing post-partum anoestrus (>120 days after parturition) and normal cyclicity (
- Effect of vitrification on the mRNA transcriptome of bovine oocytes
- Authors: N Wang; C-Y Li, H-B Zhu, H-S Hao, H-Y Wang, C-L Yan, S-J Zhao, W-H Du, D Wang, Y Liu, Y-W Pang, X-M Zhao
Abstract: Vitrification has been shown to decrease the developmental capacity of mammalian oocytes, and this is closely associated with the abnormal mRNA expressions of vitrified oocytes. However, the effect of vitrification on transcriptional machinery of oocytes examined by RNA sequencing (RNA-seq) has yet to be defined. In the present study, the mRNA transcriptomes of fresh and vitrified bovine oocytes were analysed by Smart-seq2 with the differently expressed genes determined by DEseq2 (an adjusted p-value of .05 and a minimum fold change of 2). The differentially expressed mRNAs were then searched against the Gene Ontology (GO) and Genomes (KEGG) database. Finally, the mRNA expressions of 10 candidate genes were validated using quantitative real-time PCR (qRT-PCR). Approximately 12,000 genes were detected in each sample of fresh or vitrified oocytes. Of these, the expression levels of 102 genes differed significantly in vitrified groups: 12 genes mainly involved in cell cycle, fertilization and glucose metabolism were upregulated, and 90 genes mainly involved in mitochondria, ribosomal protein, cytoskeleton, transmembrane protein, cell cycle and calcium ions were downregulated. GO analysis showed that these genes were mainly enriched in terms of membrane-bounded organelles, macromolecular complex, and intracellular part. The mRNA expression levels of 10 candidate genes selected randomly were in agreement with the results of the RNA-seq. In conclusion, our results showed that vitrification affected the mRNA transcriptome of bovine oocytes by downregulating genes, which contributed to the decreased developmental capacity of vitrified oocytes. Our findings will be useful in determining approaches to improve the efficiency of vitrified oocytes.
- Endometrial biopsy in Bos indicus beef heifers
- Authors: O Ramirez-Garzon; N Satake, RE Lyons, J Hill, MK Holland, M McGowan
Abstract: An endometrial biopsy allows for a comprehensive assessment of the uterine environment of a breeding female. Although routine in mares, devices used for endometrial biopsies are impracticable in heifers due to the size and structure of the cervix. This report describes the use of a human bronchoscopy biopsy device (Karl Storz® 10366L) for collection of endometrial biopsies in Bos indicus beef heifers. The Storz® device is smaller and thinner and enabled the collection of an endometrial biopsy in 86% of heifers (n = 44/51). The biopsied tissue was of good quality and suitable for transcriptomic assessment of the endometrium, with total RNA yield and RNA integrity number (RIN) averaging 1.3 μg (range 0.4–5.3 μg) and 7.4 (range 5.7–8.4), respectively.
- Development of an in vitro oviduct epithelial explants model for studying
sperm–oviduct binding in the buffalo
- Authors: KK Saraf; A Kumaresan, S Nayak, S Chhillar, L Sreela, S Kumar, UK Tripathi, TK Datta, TK Mohanty
Abstract: In this study, we developed an in vitro model for studying sperm–oviduct binding in the buffalo. Oviduct explants were prepared by overnight culture of epithelial cells in TCM-199 medium under 5% CO2 at 38.5 °C. Cryopreserved spermatozoa from buffalo bulls (n = 4) were incubated with the oviduct explants, and the sperm–oviduct explants complex was stained with JC-1. The effect of sperm concentration (2, 3 and 4 million), size of the oviduct explants (0.4 mm2) and time of incubation (1 hr and 4 hr) on binding index (BI—number of sperm bound to unit area of explants) was studied. No significant difference was observed in the BI among
- Comparison of the effects of Ham'sF10 and αMEM in combination with FBS or
BSA in vitrification/warming solution on quality and viability of sheep
- Authors: F Mohammadzadeh; L Safdarian, F Amidi, A Mohammadzadeh, K Mortezaee, S Mehdinejhadiani, A Sobhani, S Ghasemi, F Sargolzaei Aval
Abstract: The aim of this study was to evaluate the effects of the two types of media, namely minimum essential medium (αMEM) and Ham'sF10, supplemented with foetal bovine serum (FBS) or bovine serum albumin (BSA) in vitrification/warming solution on the quality and viability of sheep ovarian follicles. Vitrification method was applied for cryopreservation of sheep ovarian cortex using Ham'sF10 and αMEM supplemented with either BSA or FBS. There were five groups: Fresh, Ham'sF10+ BSA, Ham'sF10+ FBS, αMEM + BSA and αMEM + FBS. Samples were cultured for two weeks after warming. Viability and morphology of follicles and DNA fragmentation in follicles and in tissue stroma cells were analysed before vitrification/warming and following one and two weeks of culture. The Ham'sF10+ FBS and Ham'sF10+ BSA groups showed a significant decrease in follicular viability after one week of culture (p
- Effects of supplementation of medium with different antioxidants during in
vitro maturation of bovine oocytes on subsequent embryo production
- Authors: TC Sovernigo; PR Adona, PS Monzani, S Guemra, FDA Barros, FG Lopes, CLV Leal
Abstract: The aim of this study was to assess the effects of different antioxidants on the levels of reactive oxygen species (ROS) and glutathione (GSH) in oocytes during in vitro maturation (IVM), as well as on the production of embryos. Oocyte of slaughterhouse-derived cattle ovaries were placed in IVM with different antioxidants: quercetin (2 μM), cysteamine (100 μM), carnitine (0.5 mg/ml), vitamin C (50 μg/ml) or resveratrol (2 μM). Oocytes matured without any antioxidant supplementation were used as control. The oocytes were assessed for maturation rates and for ROS and GSH levels by fluorescence staining in 2′,7′-dichlorodihydrofluorescein diacetate and Cell Tracker Blue, respectively. Embryo production was assessed in terms of cleavage, blastocysts and hatching rates and embryo cell numbers. The results expressed in arbitrary fluorescence units showed ROS reduction (p
- Peri-conceptional under-nutrition alters the expression of TRIM28 and
ZFP57 in the endometrium and embryos during peri-implantation period in
- Authors: K Zglejc; A Franczak
Abstract: DNA methylation is maintained by the main elements of methylation complex—tripartite motif containing 28 (TRIM28) and zinc finger protein 57 (ZFP57). Previously, it was found that the activity of TRIM28 and ZFP57 determines the process of DNA methylation and preserves over-expression of genes. We hypothesized that restricted diet applied during peri-conceptional period may induce changes in the expression of methylation complex in porcine endometrium and embryos during the peri-implantation period. The aim of this study was to detect and determine the expression of TRIM28 and ZFP57 in the endometrium and embryos harvested from gilts during the peri-implantation period (days 15–16 of pregnancy) fed restricted (n = 5) or normal (n = 5) diet during peri-conceptional period. In restricted-diet-fed gilts, endometrial expression of TRIM28 and ZFP57 mRNAs was decreased in comparison with normal-diet-fed gilts (p ≤ .01), while the embryonic expression of TRIM28 and ZFP57 mRNAs was increased in restricted-diet-fed gilts (p ≤ .05). The immunofluorescence showed the presence of TRIM28 and ZFP57 in luminal epithelial (LE), glandular epithelial (GE) and stromal cells (ST) of the endometrium as well as in the embryos. Total endometrial and embryonic abundance of TRIM28 and ZFP57 proteins was significantly higher (p ≤ .05) in restricted-diet-fed gilts than in normal-diet-fed gilts. Female under-nutrition during peri-conceptional period affects the expression of two main elements of methylation complex in the endometrium and in embryos during the peri-implantation period and may have the impact on DNA methylation in these tissues.
- Bax and Bcl-2 are involved in the apoptosis induced by local testicular
heating in the boar testis
- Authors: H Xi; X Fan, Z Zhang, Y Liang, Q Li, J He
Abstract: The aim of this study was to determine whether the effect of Bax and Bcl-2 on the apoptosis of germ cells is caused by local testicular heating (42°C, 1 hr) in boar testis. The testes of three boars were exposed to 42°C for 1 hr. Three other boars were assigned as control (no heat treatment). After 6 hr of heat treatment, all boars were castrated and the testes were harvested. Immunohistochemical results showed that a redistribution of Bax was caused by heat stress, and Bcl-2 was expressed in the cytoplasm and nucleus. Western blot analyses and quantitative real-time polymerase chain reaction (QRT-PCR) showed that the protein and mRNA levels of Bax and Bcl-2 were increased after local testicular heating. The number of TUNEL-positive cells was increased in the seminiferous tubules compared with the control after local testicular heating. These results suggested that local testicular heating induced the apoptosis of germ cells by regulating the Bax and Bcl-2 protein levels.
- Metabolomic profiling in umbilical venous plasma reveals effects of
dietary rumen-protected arginine or N-carbamylglutamate supplementation in
nutrient-restricted Hu sheep during pregnancy
- Authors: L Sun; H Zhang, Y Fan, Y Guo, G Zhang, H Nie, F Wang
Abstract: Maternal nutrient restriction during pregnancy is a major problem worldwide for human and animal production. Arginine (Arg) is critical to health, growth and reproduction. N-carbamylglutamate (NCG), a key enzyme in arginine synthesis, is not extensively degraded in rumen. The aim of this study was to investigate ameliorating effects of rumen-protected arginine (RP-Arg) and NCG supplementation on dietary in undernourished Hu sheep during gestation. From day 35 to 110 of gestation, 32 Hu ewes carrying twin foetuses were randomly divided into four groups: a control (CG) group (n = 8; fed 100% National Research Council (NRC) requirements for pregnant sheep), a nutrient-restricted (RG) group (n = 8; fed 50% NRC requirements, which included 50% mineral–vitamin mixture) and two treatment (Arg and NCG) groups (n = 8; fed 50% NRC requirements supplemented with 20 g/day RP-Arg or 5 g/day NCG, which included 50% mineral–vitamin mixture). The umbilical venous plasma samples of foetus were tested by 1H-nuclear magnetic resonance. Thirty-two differential metabolites were identified, indicating altered metabolic pathways of amino acid, carbohydrate and energy, lipids and oxidative stress metabolism among the four groups. Our results demonstrate that the beneficial effect of dietary RP-Arg and NCG supplementation on mammalian reproduction is associated with complex metabolic networks.
- Effect of selenium supplementation on semen characteristics of Brazil's
- Authors: M Piagentini; DC Silva, CPF Dell'Aqua, CF Moya-Araujo, VM Codognoto, AA Ramos, E Oba
Abstract: The aim of this study was to investigate the effects of different concentrations of oral supplementation with selenium (Se) upon ram sperm parameters. Thirty rams managed in stall under intensive system were used and divided into five groups (six animals per group) as follows: control group (G1) mineral mixture supplementation without Se, group 2 (G2) mineral mixture supplemented with 5 mg/kg Se, group 3 (G3) supplemented with 10 mg/kg Se, group 4 (G4) supplemented with 15 mg/kg Se and group 5 (G5) supplemented with 20 mg/kg Se. For each group, there was an adjustment period of 14 days. The experimental period was 350 days. Every 56 days, the animals were weighed and semen samples were collected by electroejaculation. Semen analysis included volume, mass moviment, total motility, vigour, concentration and morphology. For plasmatic and acrosomal membrane integrity evaluation and mitochondrial membrane potential were used a combination of fluorescent probes. Differences between means values obtained by analysis of variance were verified by Tukey test with 5% probability. There was no statistical difference between treatment groups in relation to volume, mass moviment, total motility, vigour, concentration, plasma and acrosomal membrane integrity (p > .05). Sperm morphology was different between treatment groups, the G1 (0 mg of selenium) had the highest percentage of major defects (11.11 ± 1.11a; p
- Identification of sperm morphometric subpopulations in cooled-stored
canine sperm and its relation with sperm DNA integrity
- Authors: M Urbano; I Ortiz, J Dorado, M Hidalgo
Abstract: The aims of this study were to (i) identify different morphometric subpopulations in cooled-stored canine sperm and their patterns of distribution during cool-storage for up to 240 hr and (ii) determine whether or not morphometric sperm subpopulations (sP) are related to sperm DNA integrity. For that purpose, morphometric parameters were analysed by computer-assisted sperm analysis (CASA) and sperm DNA fragmentation (sDFi) using the sperm Halomax test. Four morphometric sperm heads subpopulations were identified: sP1 (large and rounded), sP2 (large and elongated), sP3 (small and rounded) and sP4 (small and elongated). sP1 was the most predominant subpopulation for up to 72 hr and thereafter sP3 increased progressively. sDFi increased after 48 hr of cool-storage. Although sP3 showed a positive correlation with sDFi, and both increased over time, it could not be ensured that only the sperm with fragmented DNA are accumulated in sP3. In conclusion, sP3 and DNA fragmentation increased progressively during cool-storage, becoming possible indicators of sperm damage. However, it cannot be concluded that sP3 only contains sperm with fragmented DNA.
- Impaired reproduction in Japanese Black cattle under cold environmental
- Authors: H Nabenishi; A Yamazaki
Abstract: Environmental factors such as the temperature–humidity index (THI) are known to affect reproductive parameters in cattle. Therefore, here, we examined whether there was any correlation between the THI and the reproductive performance of Japanese Black cattle by analysing the first-service conception rates of 178,492 artificially inseminated cows across 9,833 herds in south-western Japan over a 3-year period. The daily mean (±SD) THI over the study period was 63.6 ± 11.3 (range: 41.4–81.5). The calving to first artificial insemination (AI) interval was significantly negatively correlated with THI in the month of AI (r = −.75, p
- Clinical relevance of pre-ovulatory follicular temperature in
heat-stressed lactating dairy cows
- Authors: F López-Gatius; RHF Hunter
Abstract: Temperature gradients in female reproductive tissues seem to influence the success of key processes such as ovulation and fertilization. The objective of this study was to investigate whether pre-ovulatory follicles are cooler than neighbouring uterine tissue and deep rectal temperatures in lactating dairy cows under heat stress conditions. Temperatures within the pre-ovulatory follicle, on the uterine adjacent surface and 20 cm deep within rectum, were measured using fine thermistor probes within 45 min after sunrise (dawn). Cows were selected from synchronized groups for fixed-time insemination during the warm period of the year. Five cows under direct sun radiation and 11 cows in the shade were included in the study. None of the cows in the sun area ovulated within 24 hr, whereas 10 of the 11 cows in the sun area ovulated. Four of the 10 ovulating cows became pregnant. In the ovulating cows, follicular temperatures were 0.74 and 1.54°C significantly cooler than uterine surface and rectal temperatures, respectively, whereas temperatures in the uterine area were 0.80°C significantly cooler than rectal temperatures. No significant differences among temperatures were found in non-ovulating cows. Follicular size was similar for ovulating and non-ovulating cows. Environmental temperatures in the shade area were 6.4°C significantly lower than those in the sun area. Results of this study indicate that pre-ovulatory follicles are cooler than neighbouring uterine tissue and deep rectal temperatures and those temperature gradients were not found in cows suffering ovulation failure.
- Distribution of mast cells in the feline ovary in various phases of the
- Authors: P Hamouzova; P Cizek, R Novotny, A Bartoskova, F Tichy
Abstract: This study is the first description of the distribution of mast cells in various phases of the oestrous cycle in the ovary of cat. Furthermore, this is the first description in species with an induced ovulation. The aim was to describe the distribution of mast cells and variability of their numbers in the feline ovaries in different phases of the oestrous cycle. The number of mast cells in medulla ovarii was affected by the estradiol and progesterone level in the blood serum because the lowest number was detected in anoestrus when the levels of hormones were basal. Nevertheless, both high and low numbers of mast cells were found in oestrus and dioestrus. To conclude, mast cells seem to be essential for the induction of spontaneous ovulation, but they do not play the same role for ovulation itself in cats with induced ovulation.
- Supplement of autologous ooplasm into porcine somatic cell nuclear
transfer embryos does not alter embryo development
- Authors: W-J Lee; J-H Lee, R-H Jeon, S-J Jang, S-C Lee, J-S Park, S-L Lee, W-A King, G-J Rho
Abstract: Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm-transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro-fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.
- Appraisal and standardization of curvilinear velocity (VCL) cut-off values
for CASA analysis of Japanese quail (Coturnix japonica) sperm
- Authors: U Farooq; IA Malecki, M Mahmood, GB Martin
Abstract: One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut-off values of “velocity curvilinear” (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA®-CASA. The median and mean VCL values were 85 and 97 μm/s. To define the VCL cut-off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut-off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut-off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the “25%” setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two-step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 μm/s, respectively. Together, the mean from two methods suggested that, for SCA®-CASA categorization of quail sperm, sperm should be classed as “rapid” at VCL ≥160 μm/s and “slow” at VCL ≤45 μm/s.
- Localization of orexin B and receptor 2 for orexins in testicular
cytotypes of the camelid alpaca (Vicugna pacos)
- Authors: G Liguori; C Squillacioti, L Assisi, N Mirabella, E Langella, A Costagliola, A Vittoria
Abstract: The orexins A (OxA) and B (OxB) are two hypothalamic peptides involved in many physiological functions of the mammalian body. They act through the binding of two G-coupled receptors named receptor 1 (OX1) and receptor 2 (OX2) for orexins. The first receptor is specific for OxA, while the second binds both the substances with equal affinity. The orexins and the relative receptors have been traced by means of different techniques also at the periphery of the body and particularly in the adrenals, and in gastrointestinal and genital organs. Aim of this work was to investigate the presence of OxB and OX2 by means of immunohistochemistry and Western blotting analysis in the testis of the South American camelid alpaca, a species primarily breed in Chile and Ecuador and recently diffused in Europe where the quality of its wool is particularly appreciated. OxB immunoreactivity (IR) was found in the tubular compartment of the testis where spermatogonia (resting), zygotene and pachytene spermatocytes, and spermatids clearly showed differently sized and shaped cytoplasmic positive structures. OX2-IR was found both in the interstitial and tubular compartments of the testis and particularly in Leydig cells and round and elongated spermatids. Western blotting analysis of testis lysates showed the presence of a protein band whose molecular weight corresponded to that currently assigned to OX2. Such findings easily translate the hypothesis that OxB and its receptor 2 play a functional role both in the interstitial and tubular compartments of the alpaca testis.
- Protein and mRNA expression of follicle-stimulating hormone receptor and
luteinizing hormone receptor during the oestrus in the yak (Bos grunniens)
- Authors: S-d Huo; S-e Chen, R-j Long, J-t Yang, J-x Lu, R-x Zang, T-j Zhang, A. Abudureyimu, J-l Liu, G-h Zhang, Y-q Zhao, Z-r Ma
Abstract: Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have a central role in follicle growth, maturation and oestrus, but no clear pathway in the seasonal oestrus of yak (Bos grunniens) has been found. To better understand the role of FSH and LH in seasonal oestrus in the yak, six yaks were slaughtered while in oestrus, and the pineal gland, hypothalamus, pituitary gland, and gonads were collected. Using real-time PCR and immunohistochemical assays, we determined the mRNA and protein expression of the FSH and LH receptors (FSHR and LHR) in these organs. The analysis showed that the FSHR mRNA expression level was higher in the pituitary gland tissue compared with LHR (p
- Are foetal ultrasonographic and maternal blood progesterone measurements
near parturition reliable predictors of the time of birth in the domestic
- Authors: R Keiser; IM Reichler, O Balogh
Abstract: In cats, accuracy of parturition day prediction by ultrasonographic measurement of foetal structures is decreasing towards the end of gestation. Foetal measurements during the last days of pregnancy are scarce. We determined foetal biparietal, abdominal and eye diameter (BPD, AD and ED, respectively) by ultrasonography as well as maternal blood progesterone (P4) within five days of delivery to predict parturition date and calculate accuracy of prediction. Foetal BPD at birth was compared with newborn kitten head diameter (HD). Kitten HD, crown-rump length (CRL) and body weight were compared by breed and gender. Ultrasonography measurements were carried out on the day of parturition in 14 queens, and on days 62–63 after the first mating and repeated 24–72 hr later in ten other cats. Accuracy of parturition day prediction using BPD and AD was determined based on the equations of Beccaglia et al. (2008) Veterinary Research Communications, 32(Suppl 1), S99 and Garcia Mitacek et al. (2015) Theriogenology, 84, 1131. Progesterone was measured at the time of presentation and repeated 24–72 hr later if parturition did not occur. Data were analysed with linear regression, t test, Mann–Whitney U test, one-way anova and Kruskal–Wallis test. There was a moderate relationship between BPD, days before birth (DBB) and litter size. AD and DBB had a low agreement, and ED was not associated with DBB. BPD at birth was significantly related to HD. The accuracy of parturition day prediction using BPD and AD was 27–53% and 17–35%, respectively. Kitten HD was associated with body weight, and both were inversely related to litter size. Newborn biometric measurements differed by breed but not by gender. Progesterone decreased towards parturition and reached 3.18 ± 1.68 ng/ml on the day of delivery. In conclusion, close to birth, the combination of foetal ultrasonography and maternal blood P4 rather than each as a sole predictor of parturition is recommended.
- Administration of exogenous hormones in ovulatory and embryonic response
in Pelibuey sheep
- Authors: A García-Salas; C Cortez-Romero, J Salazar-Ortiz, J Arroyo-Ledezma, VM Ruíz-Vera, H Vaquera-Huerta, J Gallegos-Sánchez
Abstract: The objective of this study was to evaluate the effect of two sources of commercial porcine pituitary-derived follicle-stimulating hormone (pFSH) and pFSH—porcine Luteinizing Hormone (pLH), including equine chorionic gonadotropin (eCG), in ovulatory and embryonic response in Pelibuey sheep. Twenty-four Pelibuey sheep were used and were assigned randomly to four treatments (n = 6): (T1; 200 mg pFSH-Folltropin®); (T2; 200 mg pFSH + 300 UI eCG-Folligon®); (T3; 250 UI pFSH/pLH-Pluset®) and (T4; 250 UI pFSH/pLH + 300 UI eCG). The interval of hours from withdrawal of the device to the beginning of oestrus (BO) was lower (p
- Urine levels of luteinizing hormone as predictor of the period of
ovulation for advantage of timed-artificial insemination in murrah buffalo
- Authors: RM Selvam; D Singh, MA Akbarsha, G Archunan
Abstract: Assessment of urine levels of luteinizing hormone (LH) for predicting the reproductive status of animals is in practice. The aim of this study was to predict the period of ovulation based on the urine levels of LH for timed-artificial insemination to increase the conception rate in buffaloes, which are naturally silent-oestrous animals. Level of LH in urine was assessed using ELISA, and a cut-off LH concentration for prediction of ovulation period was obtained using receiver operating characteristic analysis. Artificial insemination was performed before- and after -positive prediction of ovulation period adopting this method, and the rates of conception were assessed. Urine LH level of 105 mIU/ml (n = 14) was derived as a cut-off concentration which predicts the ovulation period. The buffaloes in the positively predicted group (day 1 or 2) inseminated via intracervical route had an increase in the conception rate (83.33%); however, the insemination in the before-positive-prediction group resulted in poor conception rates (day 0; 16.66%) compared to that of the naturally inseminated group (day 0; 75.0%). In conclusion, the urinary LH would possibly be a fairly reliable predictor of the ovulation period. The day when cut-off LH concentration is obtained may be taken as the most favourable time for artificial insemination, so as to attain a much better rate of conception in the buffalo.
- The effect of some cryoprotectants on dromedary camel frozen-thawed semen
- Authors: DA El-Badry; RH Mohamed, HA EL-Metwally, TR Abo Al-Naga
Abstract: The cryopreserved camel semen is often associated with poor quality and fertility. This study aimed to improve the dromedary frozen semen quality by comparing the efficiency of four cryoprotectant agents (CPAs) on sperm freezability. Semen samples were collected from seven male Maghrabi camels, diluted with Shotor diluent supplemented with glycerol (Sh-G), dimethyl formamide (DMF, Sh-DF), dimethyl sulfoxide (DMSO, Sh-DS) or ethylene glycol (EG, Sh-EG), all at 6% final concentration, and the samples were subjected to cryopreservation. The results revealed the superiority of Sh-DF over Sh-G and Sh-DS in terms of post-thaw motility (55.83 ± 2.20 vs. 47.50 ± 4.33 and 45.00 ± 2.89%, respectively), sperm membrane (49.00 ± 0.58, 39.33 ± 3.33 and 42.67 ± 1.45%, respectively) and acrosomal integrities (53.00 ± 0.58, 57.33 ± 0.88 and 52.33 ± 1.45%, respectively). Sh-EG group showed the lowest post-thaw motility, plasma membrane and acrosome integrities (12.50 ± 1.44, 22.67 ± 1.45 and 30.67 ± 1.45, respectively). In conclusion, the protocols of dromedary camel semen cryopreservation could be enhanced using 6% DMF as a cryoprotectant agent.
- Effects of supplemental conjugated linoleic acids (CLA) on fresh and
post-thaw sperm quality of Holstein bulls
- Authors: R Karimi; A Towhidi, S Zeinoaldini, K Rezayazdi, M Mousavi, H Safari, F Martinez-Pastor
Abstract: This study was designed to investigate the effects of feeding-protected conjugated linoleic acid (CLA) on the semen production and sperm freezability in Holstein bulls. Twelve bulls were randomly assigned to two groups (n = 6 per group). Bulls received the normal diet (control group) or the normal diet top-dressed with 50 g of CLA (treated group) for 10 weeks. The control group received 40 g/day calcium soap of fatty acid. Fresh and post-thaw semen quality was assessed on ejaculates collected at the 0, 4, 6, 8 and 10 week of supplementation. Semen evaluations including sperm concentration, motion characteristics (subjective and computer-assisted), viability (Eosin–Nigrosin), membrane integrity (hypo-osmotic swelling test) and abnormality were conducted. Semen volume, sperm concentration and total sperm output were not affected by dietary treatment (p > .05). The proportion of spermatozoa with abnormal morphology in fresh semen significantly increased (p
- Uterine serpin (SERPINA 14) correlates negatively with cytokine production
at the foetal–maternal interface but not in the corpus luteum in
pregnant dairy heifers experimentally infected with Neospora caninum
- Authors: B Serrano-Pérez; S Almería, R Mur-Novales, I López-Helguera, I Garcia-Ispierto, JL Alabart, L Darwich, F López-Gatius
Abstract: This study examines gene expression patterns in dairy heifers experimentally infected with N. caninum during on Day 110 of pregnancy with live foetuses at euthanasia, 42 days later. The study population was constituted of four non-infected controls and three infected dams. Gene expression was determined on gamma interferon (IFNγ), (Th1 pro-inflammatory cytokine), interleukin-4 (IL4) (Th2 pro-gestation cytokine) or interleukin-10 (IL10) (T regulatory cytokine) and the serine peptidase inhibitor SERPINA14 in intercaruncular, placental, uterine lymph node (UTLN) and luteal tissue samples. Intercaruncular SERPINA14 expression was negatively correlated with IFNγ expression in cotyledon samples and with IL4 expression in UTLN. No relationships were detected between cytokine gene expression at the foetal–maternal interface and SERPINA14 expression in the luteal samples. Our findings suggest that gene expression of the uterine serpin SERPINA14 correlates negatively with the expression of Th1 and Th2 cytokines at the foetal–maternal interface but not in the corpus luteum.
- The effect of cooling to different subzero temperatures on dog sperm
- Authors: A Alcantar-Rodriguez; A Medrano
Abstract: The objective was to assess the effect of cooling to different subzero temperatures around ice formation (−5°C) on dog sperm cryosurvival and plasma membrane fluidity. Semen was centrifuged, and sperm were resuspended in a Tris-egg yolk medium (3% glycerol). Diluted sperm were cooled from 22 to 5°C, and then, a Tris-egg yolk medium containing 7% glycerol was added (final concentration of 5% glycerol and 200 × 106 cells/ml). Sperm were packaged in 0.5-ml plastic straws, and equilibration was done 16 hr at 5°C before freezing. I. Straws (n = 47) at 5°C were exposed to nitrogen vapours to determine the freezing point. II. Other straws (from different ejaculates) processed as mentioned, were further cooled to −3, −5 or −7°C and immediately rewarmed in a water bath at 37°C. Motility, plasma membrane functionality and acrosome integrity were assessed. III. Other straws (from different ejaculates) processed as mentioned were further cooled to −3 or −5°C, frozen over nitrogen vapours and stored in liquid nitrogen for one month. Straws were thawed in a water bath at 38°C for 30 s. Motility, plasma membrane functionality, plasma membrane integrity, acrosome integrity, capacitation status and plasma membrane fluidity were assessed. Ice nucleation temperature was −14.3 ± 2.05°C (mean ± SD); cooling to +5, −3, −5 and −7°C, without freezing, produces no differences on sperm quality between target temperatures; cooling to +5, −3, and −5°C produced no differences on sperm survival and plasma membrane fluidity after freeze–thawing. In conclusion, cooling of dog spermatozoa to different subzero temperatures did not improve sperm cryosurvival and had no effect on plasma membrane fluidity after thawing.
- Transporting bovine oocytes in a medium supplemented with different
macromolecules and antioxidants: Effects on nuclear and cytoplasmic
maturation and embryonic development in vitro
- Authors: M Ambrogi; PC Dall'Acqua, NAS Rocha-Frigoni, BCS Leão, GZ Mingoti
Abstract: We investigated whether supplementing the medium used to transport bovine oocytes with different macromolecules [foetal calf serum (FCS) or bovine serum albumin (BSA)] or a mixture of antioxidants (cysteine, cysteamine and catalase) affects their nuclear and cytoplasmic maturation and thereby affects their subsequent embryonic development and cryotolerance. Oocytes were transported for 6 hr in a portable incubator and then subjected to standard in vitro maturation (IVM) for 18 hr. The oocytes in the control groups were cultured (standard IVM) for 24 hr in medium containing 10% FCS (Control FCS) or 10% FCS and the antioxidant mixture (Control FCS+Antiox). The intracellular concentrations of reactive oxygen species (ROS) at the end of IVM period were lower in the oocytes subjected to simulated transport in the presence of a macromolecular supplement or the antioxidant mixture than that of the control group (FCS: 0.62 and BSA: 0.66 vs. Control FCS: 1.00, p
- Ejaculation does not contribute to the stress response
to electroejaculation in sheep
- Authors: S Abril-Sánchez; A Freitas-de-Melo, JP Damián, J Giriboni, A Villagrá-García, R Ungerfeld
Abstract: Electroejaculation procedures (EEPs) provoke stress; nevertheless, ejaculation produces physiological changes similar as those usually used to measure stress responses. The application of EEP to animals that cannot ejaculate—as ewes—may be useful to discriminate the responses induced by ejaculation from those provoked by EEP. The aim was to determine the stress response to EEP in rams and ewes. The EEPs were applied to 10 rams and 10 ewes during the non-breeding season, and the number of vocalizations, the heart rate, rectal temperature, serum cortisol concentration, biochemical and haematological parameters were measured. Overall, EEP provoked increases in cortisol concentration, glycaemia, rectal temperature and concentration of creatine kinase (all them: p
- Lack of effects of an equine chorionic gonadotropin (eCG) administration
between days 9 and 15 postpartum on reproductive performance in a Holstein
- Authors: M Freick; O Passarge, J Weber
Abstract: Recently, it has been demonstrated that administration of equine chorionic gonadotropin (eCG) in the postpartum period in dairy cows can enhance follicle growth, reduce the interval from calving to first ovulation and increase plasma estradiol concentrations, and, thus, could enhance reproductive performance in a dairy herd when administered on day 6 postpartum. The aim of this study was to investigate the effects of a single dose of eCG between days 9 and 15 postpartum on parameters of reproductive performance in dairy cows. German Holstein cows (n = 1937; primiparous cows: n = 748; pluriparous cows: n = 1189) in a commercial dairy farm were randomly assigned to three experimental groups. Animals within the group eCG received a single dose of 600 IU eCG intramuscularly (i.m.) between days 9 and 15 postpartum followed by an i.m. administration of 500 μg cloprostenol after 14 days. Those of treatment group PG received cloprostenol only between days 23 and 29 postpartum. Cows of the control group remained untreated. Starting on day 49 postpartum, cows were subjected to a Presynch-Ovsynch protocol and inseminated artificially. The impact of application time (days postpartum) of eCG on the intervals calving to first service and calving to conception was statistically not significant. Outcomes of reproductive performance (i.e. first service conception rate, proportion of pregnant cows until 100 and 150 days in milk [DIM], number of inseminations until 150 DIM, calving to first service interval and calving to conception interval) did not differ significantly between treatment group eCG and group PG compared to control group. Regarding postpartum eCG administration, significant interactions between treatment and parity, season, milk yield, and early puerperal disorders, respectively, could not be shown. In conclusion, an eCG treatment of dairy cows between days 9 and 15 postpartum to increase reproductive performance cannot be recommended under the given circumstances.
- Dose rates of antimicrobial substances in boar semen preservation—time
to establish new protocols
- Authors: M Schulze; M Grobbel, A Riesenbeck, S Brüning, J Schaefer, M Jung, R Grossfeld
Abstract: To achieve a standardized number of spermatozoa in the final AI dose, varying amounts of extender fluid with a fixed concentration of antimicrobial substances are currently added to boar ejaculates. This practice ignores the different degrees of dilution of the antimicrobials in the end product. In calculating the final concentration of gentamicin in AI doses from 27,538 processed boar ejaculates, we demonstrated varying gentamicin concentrations in the resultant extended boar semen samples. The median concentration was 220.37 mg/L. In 25 of the samples (0.09%), the gentamicin concentration fell below 5 mg/L, which is close to or below the epidemiological cut-off value for many bacteria. We calculated the minimum inhibitory concentration of gentamicin for bacteria isolated from raw and extended ejaculates. Five of the isolates from extended ejaculates exceeded the maximum test concentration of 512 mg/L. As a result, we are presenting an alternative method of boar semen preservation whereby a particular combination of gentamicin concentrate and antibiotic-free extender is incorporated that standardizes the antibiotic concentration in the diluted semen. The addition of standardized antibiotic concentrations did not negatively affect sperm quality when compared to the use of ready-to-use extenders. In conclusion, an end volume-based and standardized addition of gentamicin to boar ejaculates can be a helpful alternative to prevent insufficient dosage of antibiotics in liquid preserved boar semen without affecting semen quality.
- Issue Information
- Pages: 1 - 2
- Undifferentiated embryonic cell transcription factor 1 (UTF1) and deleted
in azoospermia-like (DAZL) expression in the testes of donkeys
- Authors: YS Lee; HJ Jung, MJ Yoon
Pages: 264 - 269
Abstract: Putative markers for each specific germ cell stage can be a useful tool to study the fate and functions of these cells. Undifferentiated embryonic cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans, rats and horses. The deleted in azoospermia-like (DAZL) protein is also expressed by differentiated spermatogonia and primary spermatocytes in several species. However, whether the expression patterns of these molecular markers are identical and applicable to donkeys remains to be elucidated. The objective of this study was to investigate the expression patterns of UTF1 and DAZL in donkey testicular tissue, using immunohistochemistry (IHC). Testicular samples were collected from routine field castration of donkeys in Korea. The reproductive stages (pre- or post-puberty) of the testes were determined from the morphological characteristics of cross-sections of the seminiferous tubules. For IHC, the UTF1 and DAZL primary antibodies were diluted at 1:100 and 1:200, respectively. The immunolabelling revealed that UTF1 was expressed in approximately 50% of spermatogonia in the pre-pubertal stage, whereas its expression was limited to an early subset of spermatogonia in the post-pubertal stage. DAZL was expressed in some, but not all, spermatogonia in the pre-pubertal spermatogonia, and interestingly, its expression was also observed in spermatogonia and primary spermatocytes in the post-pubertal stage. Co-immunolabelling of the germ cells with both UTF1 and DAZL revealed three types of protein expression patterns at both reproductive stages, namely UTF1 only, DAZL only and both UTF1 and DAZL. These protein molecules were not expressed in Sertoli and Leydig cells. In conclusion, a co-immunolabelling system with UTF1 and DAZL antibodies may be used to identify undifferentiated (UTF1 only), differentiating (UTF1 and DAZL), and differentiated spermatogonia (DAZL only) in donkey testes.
- Epigallocatechin-3-gallate (EGCG) and green tea polyphenols do not improve
stallion semen parameters during cooling at 4°C
- Authors: D Bucci; M Spinaci, B Mislei, B Gadani, G Rizzato, CC Love, C Tamanini, G Galeati, G Mari
Pages: 270 - 277
Abstract: Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24–72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin-3-gallate (EGCG) at 20, 60 and 120 μm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.
- Lipid and protein oxidation levels in spermatozoa and seminal plasma of
Asian Elephants (Elephas maximus) and their relationship with semen
- Authors: S Satitmanwiwat; K Promthep, K Buranaamnuay, S Mahasawangkul, K Saikhun
Pages: 283 - 288
Abstract: Peroxidation damage to spermatozoa and seminal plasma has an important role in sperm quality. Thus, the objective of this study was to determine the levels of lipid and protein oxidation in spermatozoa and seminal plasma of Asian elephants (Elephas maximus) with varying percentage of progressive motility. Lipid and protein oxidation was measured by the thiobarbituric acid-reactive species (TBARS) assay and the 2, 4-dinitrophenylhydrazine (DNPH) carbonyl groups assay, respectively. Fresh semen samples were collected from Asian elephants and classified according to the percentage of motile spermatozoa into good (>60%) and poor (≤20%) motility. Results revealed that seminal plasma malondialdehyde (MDA) and seminal plasma protein carbonyls (PCs) were significantly higher in poor motility than in good motility (p
- Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm
- Authors: JDA Losano; DSR Angrimani, A Dalmazzo, BR Rui, MM Brito, CM Mendes, GKV Kawai, CI Vannucchi, MEOA Assumpção, VH Barnabe, M Nichi
Pages: 289 - 297
Abstract: Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p
- Reproductive performance of breeder quails fed diets supplemented with
L-cysteine-coated iron oxide nanoparticles
- Authors: H Mohammadi; A Farzinpour, A Vaziry
Pages: 298 - 304
Abstract: The objective of this study was to investigate the effects of L-cysteine-coated iron oxide nanoparticles on reproductive performance in breeder quails. The five treatment diets consisted of (i) negative control diet not supplemented with iron, (ii) positive control diet supplemented with 60 mg/kg of Fe3O4 and (iii) experimental diets supplemented with 0.6, 6 and 60 mg/kg of L-cysteine-coated iron oxide nanoparticles. A total of 100 seven-day-old quail chicks were weighed and randomly placed to five groups of five replicate cages. Four quails (one male and three females) were raised in each cage (50 × 15 × 17 cm). Egg production, feed consumption and egg weight were recorded daily and calculated on a hen per day basis. Egg components, fertility, hatchability and day-old chicks hatched from their eggs were measured at the end of the experiment. The percentage of egg production and egg mass of the 6 mg/kg Fe3O4-Cys NPs group were significantly higher than those of the control groups. Throughout the experimental period, the highest weekly egg weight was recorded for the 60 mg/kg Fe3O4-Cys NPs group. Fertility was improved by diet supplemented with iron, both FeSO4 and Fe3O4-Cys NPs. The breeder fed Fe3O4-Cys NPs had the highest day-old chicks weight. The results of this study showed that Fe3O4 nanoparticles that were coated by L-cysteine could improve availability and utilization of iron in diet. Finally, it was proposed that Fe3O4-Cys NPs could be used as feed additives in quails.
- Effect of sperm pretreatment with glutathione and membrane destabilizing
agents lysolecithin and Triton X-100, on the efficiency of bovine
intracytoplasmic sperm injection
- Authors: F Zambrano; L Aguila, ME Arias, R Sanchez, R Felmer
Pages: 305 - 311
Abstract: Intracytoplasmic sperm injection (ICSI) is an assisted reproduction tool with several applications. Its effectiveness in bovines is lower than that in other species, mainly because of difficulties in the decondensation of the sperm nucleus after injection, and the presence of the acrosome and the plasma membrane which remain intact in this procedure. In this study, we assessed the effect of lysolecithin (LL) and Triton X-100 (TX), in combination with glutathione (GSH) as sperm pretreatments prior to ICSI. The GSH-LL and GSH-TX groups showed 0% of spermatozoa with intact membrane (SYBR 14+/PI), in comparison with the control (63.3%) and GSH (65.7%) groups. The proportions of spermatozoa with damaged acrosome membrane in the GSH-LL, GSH-TX, GSH and control groups were 46%, 35.9%, 10.5% and 7.5%, respectively. Sperm chromatin decondensation analysis showed that the groups incubated for 3 hr with GSH presented greater decondensation (p
- Gonad histology and serum 11-KT profile during the annual reproductive
cycle in sterlet sturgeon adult males, Acipenser ruthenus
- Authors: A Golpour; C Broquard, S Milla, H Dadras, AR Baloch, T Saito, M Pšenička
Pages: 319 - 326
Abstract: The aim of this study was to assess monthly testicular development in the cultured breeding stock of sterlet, Acipenser ruthenus, using histological and serum sex steroid changes. Testicular development in the adult male was examined monthly and showed four distinct phases including resting, pre-spawning, spawning and post-spawning. Also, seasonal changes of the testes were described according to its variations in gonadosomatic index (GSI) during different phases of testicular development. Using histology, we identified continuous spermatogenesis and asynchronous gonad development pattern in the testes of male sterlet, which shows that regulation of annual gonadal cycle is influenced by season. Results also showed variation in the GSI value and number of spermatogenic cells according to each season during annual cycle of gonad, as the highest value of GSI was recorded during spawning phase (spring; March-May). Hormonal profiles of 11-ketotestosterone (11-KT) showed peak, which indicated a seasonal pattern of gonadal development. The 11-KT concentration increased considerably during the spermatogenesis (pre-spawning phase) and remained quite high throughout the pre-spermiation period. In the final phase of testicular development (spawning phase), the 11-KT markedly dropped. This study undertook an examination of complete reproductive development in cultured sterlet sturgeon to provide a valuable guide for the future sterlet studies, and allows comparison of reproductive development between sturgeon species.
- Establishment and characterization of a coculture system of equine
endometrial epithelial and stromal cells
- Authors: L Lapko; D Böttcher, T Theuß, J Klug, H-A Schoon
Pages: 327 - 334
Abstract: To investigate the equine endometrium as close to the in vivo situation as possible, we established a coculture system for epithelial and stromal cells (ECs/SCs). ECs and SCs were isolated from nine endometrial tissue specimens. ECs obtained as glandular formations were cultivated on one side of the semipermeable membrane of a Millicell® insert. After 2 days, SCs (2 × 104 cells/membrane) were seeded onto the other side of the same membrane. During cocultivation, the low serum containing culture medium (Theuß et al., 2010) was supplemented with different concentrations and combinations of 17β-estradiol (2.0–3.0 pg/ml medium) and progesterone (0.5–15.0 ng/ml medium). Once the cocultures formed continuous cell layers as determined by phase-contrast microscopy, the membranes were fixed and processed for light microscopical examination. Cytokeratin 19, steroid hormone receptors and the uterine proteins uteroglobin and calbindinD9k were detected using immunocytochemistry to determine the degree of culture purity and functional cellular differentiation. The culture purity of the EC layer averaged ≥95%. Uteroglobin and calbindinD9k were consistently expressed in ECs, while hormone receptors were predominantly absent in both cell populations. An explicit cytomorphological epithelial differentiation with formation of round-oval to polygonal cell forms was encountered in ≤50% of all ECs and independent of supplemented steroids. Based on the findings altogether, and despite the partly absent congruence to the in situ prerequisites, we established a standardized and reproducible coculture system, which offers a basic approach for studies of physiologic and pathophysiologic issues in the mare.
- Testosterone serum profile, semen characteristics and testicular biometry
of Mangalarga Marchador stallions in a tropical environment
- Authors: B Waddington; JM Penitente-Filho, JGS Neves, RO Pinho, AY Chaya, PP Maitan, CO Silveira, MG Neves, SEF Guimarães, GR Carvalho, JD Guimarães
Pages: 335 - 343
Abstract: This study was conducted to characterize the daily profile of testosterone secretion and its mean concentrations in the four seasons as well as to evaluate the semen characteristics and testicular biometry of Mangalarga Marchador stallions throughout the year in a tropical region. Three stallions were submitted to semen collections and evaluation of testicular biometry every 14 days along a year. Blood samples were collected once at the middle of each season, in a 20-min interval during 24 hr in order to evaluate the testosterone secretion profiles among seasons. Testosterone concentrations along the day were higher at the beginning of the afternoon (from 12:00 to 15:00 hr), but a circadian secretion was not clearly observed. Mean testosterone concentrations did not differ among seasons (p > .05), but a pattern of secretion along the day showed variations with higher concentrations in the afternoon during the winter. Ejaculate volume was higher during summer; however, sperm motility decreased in summer and spring. Total sperm in ejaculate, sperm morphology and testicular biometry kept constant along the year showing no differences among the seasons. The results demonstrated that in a tropical region, reproductive aspects of stallions did not show a clearly defined seasonal variation, and months of autumn and winter were not unsuitable for reproduction of the males.
- Short communication: Progressive motility of frozen–thawed canine semen
is highest five minutes after thawing
- Authors: S Karger; B Geiser, M Grau, W Heuwieser, SP Arlt
Pages: 350 - 352
Abstract: Progressive motility is usually estimated by visual inspection using a light contrast microscope at X 100 immediately after semen collection or immediately after thawing frozen semen. Standard operating procedures have never been established for this test. The objective of this experiment was to examine time-dependent changes of motility after thawing cryopreserved canine semen. Semen of 35 dogs was collected, and volume, concentration, progressive motility, morphology, membrane integrity and HOS test were evaluated. For cryopreservation, CaniPRO® Freeze A&B was used. Semen was thawed and diluted using CaniPRO® culture medium. After thawing, semen was evaluated as before. In addition, every sample was evaluated for progressively motile sperm cells 0, 5, 20 and 60 min after thawing. Progressive semen motility was significantly highest five minutes after thawing.
- Efficiency of uterine fluid cytology in the diagnosis of subclinical
endometritis in the water buffalo (Bubalus bubalis)
- Authors: SC Gahlot; S Kumar, A Kumaresan, S Chand, RK Baithalu, S Lathika, TK Patbandha, SS Lathwal, TK Mohanty
Abstract: This study compared endometrial cytology vis-a-vis uterine fluid cytology for assessment of uterine health in clinically normal and subclinical endometritis (SE)-affected buffaloes. Uterine fluid samples and endometrial samples were collected from the buffaloes (n = 38) at oestrus using blue sheath and cytobrush, respectively. The smears were stained with Field stain for 3 minutes, and a minimum of 400 cells were counted in each smear for determination of the percentage of polymorphonuclear (PMN) leucocyte. The incidence of subclinical endometritis, based on the cytobrush cytology, was 23.08%. The correlation between cytobrush cytology with uterine fluid cytology was positive and significant (r = .37; p = .02). The ratio of PMN leucocyte in cytobrush cytology to uterine fluid cytology was 1:2.4. ROC analysis revealed that the threshold value of 6.16% PMN leucocyte in uterine fluid cytology showed a diagnostic sensitivity and specificity of 100% in differentiating normal from SE-affected buffaloes. In conclusion, collection of uterine fluid was easier compared to collection of endometrial samples using cytobrush and the percentage of PMN leucocyte in uterine fluid cytology can be used as a tool for diagnosis of subclinical endometritis in buffaloes.
- Effect of testicular tissue lysate on developmental competence of porcine
oocytes matured and fertilized in vitro
- Authors: AK Singh; S Naskar, B Saikia, Y Vashi, S Gupta, S Banik, MK Tamuli, V Pande, DK Sarma, SK Dhara
Pages: 183 - 188
Abstract: The objective of the present study was to investigate the effect of testicular tissue lysate (TTL) on developmental competence of germinal vesicle (GV) stage porcine oocytes. Two types of TTL were prepared through repeated freeze–thaw in liquid nitrogen, one from whole testicular tissue (wTTL) and other from either of four different sections of testes, namely just beneath the tunica albuginea (TA), from the transitional area between the seminiferous cord/tubules and the mediastinum testis (TR) and from the intermediate area (parenchymal tissue origin) and CE (cauda epididymis origin). The whole or section-wise TTL treatments were given for 44 hr during in vitro maturation (IVM). Oocyte maturation was done in either of the two media, namely defined (high-performance basic medium for porcine oocyte maturation, commercially available) and serum containing (TCM199). After maturation, oocytes were co-incubated with fresh spermatozoa for 6 hr and then transferred to embryo culture media. Treatment of GV stage oocytes with wTTL (1 mg/ml) increased the cleavage and morula percentage rate (69.23 ± 6.23 and 48.15 ± 6.77, respectively) than that of their control (58.33 ± 8.08 and 32.54 ± 5.53, respectively) in defined media, and in serum-containing media, cleavage and morula percentage rate were almost equal in both treatment (54.56 ± 7.79 and 34.70 ± 6.78, respectively) and control (59.52 ± 8.21 and 38.52 ± 6.54, respectively). However, effect of wTTL was not significant. In case of section-wise TTL supplements, TR section significantly (p
- Nasal immunization with inhibin DNA vaccine delivered by attenuated
Salmonella choleraesuis for improving ovarian responses and fertility in
- Authors: Q Liu; ZU Rehman, JJ Liu, L Han, XR Liu, LG Yang
Pages: 189 - 194
Abstract: This study was conducted to determine the effect of immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis on ovarian responses and fertility in cross-bred buffaloes. A total of 134 cross-bred buffaloes were divided into four groups: groups T1 (n = 34), T2 (n = 35) and T3 (n = 31) were nasal immunized twice a day with 10 ml of 1 × 1010 CFU/ml of the C501 (pVAX-asd-IS) vaccine for 5, 3 and 1 day, respectively. Group C (n = 34) was nasal immunized with 10 ml PBS for 5 days. All animals were immunized twice with an interval of 14 days and administered with 200 μg of a GnRH analogue on day 28, 0.5 mg PGF2α on day 35 and 200 μg of the same GnRH analogue on day 37. TAI was performed at 18 and 24 hr after the second GnRH treatment. Fourteen days after primary immunization, C501 (pVAX-asd-IS) elicited significant immune responses, and anti-inhibin IgG antibody titres in group T1 were significantly higher (p
- Antisperm antibodies in repeat-breeding cows: Frequency, detection and
validation of threshold levels employing sperm immobilization, sperm
agglutination and immunoperoxidase assay
- Authors: SK Srivastava; S Shinde, SK Singh, S Mehrotra, MR Verma, AK Singh, S Nandi, N Srivastava, SK Singh, TK Goswami, SK Bhure, H Kumar, SK Ghosh
Pages: 195 - 202
Abstract: Antisperm antibodies have been found in repeat-breeding(RB) cows, and those causing agglutination and/or immobilization of sperm are considered to be closely related to unexplained infertility. However, a standard protocol for identifying antisperm antibodies (ASA) in cattle is not validated. Therefore, an investigation was undertaken to evaluate sperm immobilization (SIT), sperm agglutination (SAT) and immunoperoxidase (IPT)assays for detection of ASA in serum and their respective threshold levels for confirmation. Animals (heifers, normally breeding, repeat-breeding and pregnant animals) that were free from IBR, brucellosis and uterine infections (screened by clinical examination) were included in the study. Sperm agglutinating, sperm immobilizing and antisperm antibodies evaluated by respective assay were significantly higher (p
- miRNA and piRNA expression profiles of breeder cock testes detected by
- Authors: SR Wu; W Guo, YL Li, XC Ren, XY Lei, XY Li, JH Yao, XJ Yang
Pages: 203 - 213
Abstract: miRNAs are small non-coding regulatory RNAs that play key roles in diverse biological processes. In this study, we used the Solexa sequencing technique to profile miRNAs in breeder cock testes to illustrate their functions. A total of 663 co-expressed miRNAs and 3,180 co-expressed piRNAs were detected in three libraries. Based on Mir-X™ miRNA qRT-PCR, three miRNAs representing low, medium and high expression levels according to the sequencing results were selected randomly to validate the miRNAs' expression profiles. Results suggested that the miRNA expression profiles data could represent actual miRNA expression levels. Moreover, target genes prediction of the co-expressed miRNAs and further Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed, which revealed that some candidate miRNAs were involved in the regulation of the spermatogenesis process, spermatozoa function and testicular metabolism. In conclusion, we provided a useful resource for further elucidation of the miRNAs' regulatory role in spermatogenesis, contributing to a preliminary database for functional and molecular mechanistic studies in testicular metabolism, spermatogenesis and other testes functions.
- The impact of cross-breeding Egyptian and Italian buffalo on reproductive
and productive performance under a subtropical environment
- Authors: MAF Nasr
Pages: 214 - 220
Abstract: The aim of this study was to investigate the reproductive and productive performance of pure Egyptian (PE) buffaloes and their crosses with Italian buffaloes. In this study, 2969 dairy buffaloes were used (1599 PE; 615 F1 crosses, 50% PE and 50% Italian buffaloes; and 755 backcross [BC], 75% PE and 25% Italian buffaloes). When compared to PE, the BC and F1 had a significantly lower incidence of calving difficulty (odds ratio [OR] = 0.18, p
- Estimation of endogenous levels of osteopontin, total antioxidant capacity
and malondialdehyde in seminal plasma: Application for fertility
assessment in buffalo (Bubalus bubalis) bulls
- Authors: P Kumar; M Saini, D Kumar, A Bharadwaj, PS Yadav
Pages: 221 - 226
Abstract: This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high-fertile (conception rate>50%) and subfertile bulls (conception rate
- First case of sterility associated with sex chromosomal abnormalities in a
- Authors: J Dorado; G Anaya, M Bugno-Poniewierska, A Molina, A Mendez-Sanchez, I Ortiz, M Moreno-Millán, M Hidalgo, P Peral García, S Demyda-Peyrás
Pages: 227 - 234
Abstract: Chromosomal abnormalities are one of the main causes of genetic infertility in horses. Currently, their detection rate is rising due to the use of new diagnostic tools employing molecular markers linked to the sex chromosome pair. Despite genetic similarities, there are no previous reports of sterility associated with chromosomal abnormalities in the domestic donkey (Equus asinus). Hereby, we determined the presence of a chromosomal mosaicism in a female donkey with reproductive problems using molecular methodologies developed for horses. A two-and-a-half-year-old jenny characterized by morphological abnormalities of the reproductive tract was cytogenetically analysed using conventional and fluorescent techniques and a group of microsatellite markers (short tandem repeat, STR). At the same time, five ultrasound measures of the reproductive tract were taken and compared with eight contemporary jennies of the same breed. After slaughter, morphological examinations showed that the case study had a blind vaginal vestibule defining an empty pouch that covered the entrance of the cervical os. Histopathological studies demonstrated that this abnormal structure was compatible with a remnant hymen. Molecular markers, STR and fluorescent in situ hybridization determinations revealed that the animal was a 62, XX/61,X mosaic and, therefore, the first case of chromosomal abnormalities in the sex pair reported in donkeys.
- Isolation, proliferation and characterization of endometrial canine stem
- Authors: V De Cesaris; S Grolli, C Bresciani, V Conti, G Basini, E Parmigiani, E Bigliardi
Pages: 235 - 242
Abstract: In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT-PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue-derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue-derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue-derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow-derived MSC in dog.
- Bovine ovarian stem cells differentiate into germ cells and oocyte-like
structures after culture in vitro
- Authors: GB Souza; JJN Costa, EV Cunha, JRS Passos, RP Ribeiro, MVA Saraiva, R Hurk, JRV Silva
Pages: 243 - 250
Abstract: Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte-like structures. The ovarian stem cells were isolated and cultured in α-MEM+ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p
- Environmental and maternal factors associated with gross placental
morphology in dairy cattle
- Authors: MM Kamal; M Van Eetvelde, L Vandaele, G Opsomer
Pages: 251 - 256
Abstract: This article reports on a study of gross placental morphology of 282 expelled placentas from 89 primi- and 193 multiparous Holstein dams immediately after calving and examines associations with environmental factors such as typical herd features and season of calving, and maternal factors such as age at calving, level of milk yield at conception and cumulative amount of milk produced during gestation. The highest correlation between calf measurements and placental characteristics was found between the weight of the calf and the total cotyledonary surface (r = .643; p
- Evaluation of epididymis storage temperature and cryopreservation
conditions for improved mitochondrial membrane potential, membrane
integrity, sperm motility and in vitro fertilization in bovine epididymal
- Authors: M Nichi; T Rijsselaere, JDA Losano, DSR Angrimani, GKV Kawai, IGF Goovaerts, A Van Soom, VH Barnabe, JBP De Clercq, PEJ Bols
Pages: 257 - 263
Abstract: The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post-mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty-six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2–3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer-assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre-freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post-thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.
- The onset of puberty in Cameroon Dwarf goats kept as pets in northwestern
- Authors: D Đuričić; S Vince, H Valpotić, I Žura Žaja, R Turk, M Lojkić, I Getz, V Berta, M Samardžija
Pages: 278 - 282
Abstract: The aim of this study was to determine onset of puberty in Cameroon Dwarf goats (CDGs) kept as pets in northwestern Croatia by determining progesterone (PGS) and insulin-like growth factor-I (IGF-I) levels in the blood by RIA methods. The first cyclic ovarian activity was estimated according to hormone profiles as determined in CDG in a moderate climate environment. Sixteen female CDG kids were kept in stables with access to pasture which provided space for exercise. The goat kids born in winter (December–January–February), spring (March–April–May), summer (June–July–August) and autumn (September–October–November) were assigned into four groups according to the season of the year of birth (n = 4 in each group). At 75 days of age at the initiation of the study, they weighed between 3.2 and 5.1 kg (4.24 ± 0.53 kg). The onset of ovulatory activity was determined by PGS and IGF-I serum concentrations every 10 days starting from 75 days to 155 days of age. The onset of puberty in CDG kids occurred on average at 141.15 ± 2.66 days of age, but varied depending on the season of birth. All CDG kids born during summer were in heat by 155 days of age. In the other groups (kids born in autumn, winter or spring), one goat in each group was not in heat. Changes in blood serum PGS and IGF-I concentrations during prepubertal and pubertal periods could aid in the evaluation of reproductive status and determination of the onset of puberty in CDG during all seasons of the year in a moderate climate region.
- Assessment of spermatozoa in fertile alpaca (Vicugna pacos) males: Study
of sperm head morphometry using a nonautomated digital method and sperm
morphology based on strict criteria
- Authors: D Evangelista-Vargas; S Evangelista-Vargas, M Valdivia, A Santiani
Pages: 312 - 318
Abstract: Although computer-assisted systems for sperm morphometry and morphological analysis are important tools in the study of male fertility, their use in extensive systems in alpacas is limited by factors such as the expense of equipment and the high altitudes of the Andean region. The objectives of this study were to evaluate alpaca sperm head morphometry using a nonautomated digital method and determine the frequency of sperm abnormalities based on strict criteria for sperm morphology in fertile male alpacas. Ejaculates (n = 15) from seven alpacas were collected, and sperm smears stained with modified Papanicolaou were processed. For morphometric analysis, 3,000 sperm (200 cells/sample) images were captured at 400× magnification and Quick Photo MICRO 3.0 software was used for manual measurement of basic (sperm head length, width, perimeter and area) and derived variables (ellipticity, shape factor, elongation and regularity). For morphology assessment, smears were observed at 1000× magnification according to WHO and strict criteria. Average morphometric parameters were length 5.48 μm, width 2.99 μm, perimeter 13.62 μm, area 12.43 μm2, ellipticity 1.86, shape factor 1.20, elongation 0.29 and regularity 1.05. Significant between-individual and within-individual differences were found in morphometric parameters. Based on morphometric study, sperm heads were classified as elliptical or normal (49%), long (18%), short (2%), pyriform (12%), round (9%), large (6%) and small (4%). Morphological analysis found no additional sperm head defects in 49% of normal sperm obtained by morphometry, although a 4% incidence of neck/mid-piece defects and a 16% incidence of principal-piece defects were found. We conclude that sperm head morphometry assessment in fertile alpacas using a nonautomated digital method is feasible, and that defects in sperm heads constitute the main morphological alteration (>50% of the sperm population), based on WHO and strict criteria.
- A new device to inseminate cows at the base of the uterine horns
- Authors: O García-Peña; R Rangel-Santos, R Rodríguez-De Lara, CA Apodaca-Sarabia, E Maldonado-Simán
Pages: 344 - 349
Abstract: A new device (Chapingo device) to deposit semen at the base of the uterine horns of cattle was developed at Universidad Autonoma Chapingo, Mexico. Nine Holstein heifers were inseminated by transvaginal laparoscopy, using a laparoscope for cattle and the Chapingo device. A dose of sexed semen (2.1 × 106 spermatozoa) was deposited at the base of the uterine horn ipsilateral to the ovary where the preovulatory follicle was identified. Insemination was achieved in all the heifers, taking on average 13.7 ± 3.1 min per animal. In all cases, it was possible to see both ovaries, the base of the uterine horns and the oviducts. After the procedure, none of the heifers showed any type of complications such as haemorrhage, adhesions or trauma. On days 21 and 22 after insemination, four of the nine heifers (44.4%) returned into oestrus; on day 30 after insemination, one heifer was found to be pregnant by ultrasound. The results show the feasibility of generating pregnancies by transvaginal laparoscopy in heifers inseminated with sexed semen.