- Expression of Perilipin 2 (PLIN2) in Porcine Oocytes During Maturation
- Authors: RN Zhang; XW Fu, BY Jia, C Liu, KR Cheng, SE Zhu
Pages: n/a - n/a
Abstract: Perilipins have been reported to limit the interaction of lipases with neutral lipids within the droplets, thereby regulating neutral lipid accumulation and utilization. This study aimed to identify the location and expression of PLIN1 and PLIN2 in porcine oocytes during maturation. Quantitative real‐time polymerase chain reaction (qRT‐PCR), immunostaining and Western blot methods were used to characterize the expression and distribution patterns of PLIN1 and PLIN2 in porcine oocytes. The results showed that PLIN1 was not detectable in porcine oocytes. PLIN2 and BODIPY 493/503‐detected neutral lipid droplets appeared identical distribution patterns and extensive colocalization in both GV and MII porcine oocytes. PLIN2 protein expression was higher in GV oocytes than that in MII oocytes (p
- Molecular Characterization of Oxytocin Receptor Gene in Water Buffalo
- Authors: N Arunmozhi; SK Singh, T Sarath, SK Agarwal, A Doiphode, U Shankar
Pages: n/a - n/a
Abstract: Buffaloes are known for their productivity as compared to average yielding cows due to higher fat percentage, better feed conversion ability and disease resistance. On the other hand, the reproductive performances of buffaloes are often considered as poor owing to late sexual maturity, weak/silent oestrus, repeat breeder and prolonged intercalving interval. The study of cascade of events during oestrus and oestrous cycle can be useful for the improvement of reproductive efficiency of buffaloes. More precisely, the hormonal changes initiated at the molecular level within the animal determine the reproductive nature of the species. Nucleotide/protein sequence analysis serves as a vital tool in analysing the binding of the hormones for their effect or functions. In this study, we have reported cloning and characterization of the complete coding (cDNA) sequence of oxytocin receptor gene (OXTR) in buffaloes. Buffalo OXTR gene contains an uninterrupted ORF of 1176 nucleotides corresponding to an inferred polypeptide length of 391 amino acids (aa). The molecular weight of the deduced aa sequence was found to be 43 kDa with an isoelectric point of 9.253 and 16.328 charge at pH 7.0. The deduced protein sequence consists of 38 strongly basic (+) (K,R), 22 strongly acidic (−) (D,E), 186 hydrophobic (A, I, L, F, W, V) and 95 Polar (N, C, Q, S, T, Y) aa. Results indicated that aspartate (D) at aa position 85 and D, R and C at aa positions 136, 137 and 138, respectively, are conserved in buffaloes. The buffalo OXTR gene shared a per cent similarity ranging from 84.7 to 98.1 and 88.5 to 97.7 at nucleotide and deduced aa sequence levels, respectively, with that of other species. Phylogram constructed on the basis of either nucleotide or deduced aa sequences of buffalo OXTR gene showed that buffalo, cattle and sheep have diverged from human and swine and formed a separate clad. The buffalo sequence has shown maximum similarity and closeness with cattle followed by sheep both at nucleotide and at aa level.
- Positive Effects of Melatonin Treatment on the Reproductive Performance of
Young Border Leicester Rams Mated to Merino Ewes in Spring: Preliminary
- Authors: DO Kleemann; JM Kelly, LJ Arney, IL Farley, AJ Tilbrook, SK Walker
Pages: n/a - n/a
Abstract: Poor reproductive performance of Merino ewe flocks when mated to Border Leicester rams during spring may be due to seasonality of the Border Leicester breed. Two approaches were taken to test this assumption. Six young (12 months old) or six mixed‐age (12, 24 and ≥36 months old) Border Leicester rams were either treated or not treated with melatonin implants (2 × 2 design) 6 weeks before the four groups of rams were each put with approximately 300 Merino ewes for an 8‐week mating period. Implants were inserted in early September (experiment 1). The second approach was to yard or not yard ewes and mixed‐age rams on several occasions during the first 3 weeks of the mating period (experiment 2). Pregnancy rate and twinning percentage were assessed by ultrasonography. In experiment 1, melatonin treatment in young rams increased (p 0.05) for ewes mated to either melatonin or non‐melatonin‐treated young rams (36.8% vs 40.0%, respectively), whereas melatonin significantly improved (p
- The Importance of Having Zinc During In Vitro Maturation of Cattle
Cumulus–Oocyte Complex: Role of Cumulus Cells
- Authors: JM Anchordoquy; JP Anchordoquy, MA Sirini, SJ Picco, P Peral‐García, CC Furnus
Pages: n/a - n/a
Abstract: The aim of this study was to investigate the influence of zinc (Zn) on the health of cumulus–oocyte complex (COC) during in vitro maturation (IVM). Experiments were designed to evaluate the effect of Zn added to IVM medium on: DNA integrity, apoptosis, cumulus expansion and superoxide dismutase (SOD) activity of cumulus cells (CC). Also, role of CC on Zn transport during IVM was evaluated on oocyte developmental capacity. DNA damage and early apoptosis were higher in CC matured with 0 μg/ml Zn compared with 0.7, 1.1 and 1.5 μg/ml Zn (p 0.05). Superoxide dismutase activity was higher in COC matured with 1.5 μg/ml Zn than with 0 μg/ml Zn (p 0.05). Blastocyst rates were significantly higher in COC than in DO + CC and DO with the addition of 1.5 μg/ml Zn during IVM (p
- Inhibition of Angiotensin‐Converting Enzyme Increases Oestradiol
Production in Ewes Submitted to Oestrous Synchronization Protocol
- Authors: AS Costa; AS Junior, GEN Viana, MCS Muratori, AM Reis, APR Costa
Pages: n/a - n/a
Abstract: This study aimed at evaluating the effects of angiotensin‐converting enzyme inhibitor (enalapril) and angiotensin II antagonist (valsartan) on the oestradiol and progesterone production in ewes submitted to oestrous synchronization protocol. The animals were weighed and randomly divided into three groups (n = 7). A pre‐experiment conducted to verify the effectiveness and toxicity of enalapril (0.5 mg/kg LW) and valsartan (2.2 mg/kg LW) showed that, in the doses used, these drugs were effective in reducing blood pressure without producing toxic effects. In the experiment, all animals were subjected to oestrous synchronization protocol during 12 days. On D10, D11 and D12, animals received saline, enalapril or valsartan (same doses of the pre‐experiment), according to the group randomly divided. The hormonal analysis showed an increase in oestradiol on the last day of the protocol (D12) in animals that received enalapril (p
- First Description of Scrotal Testicles in a Dog Affected by 78, XX
Testicular Disorder of Sex Development
- Authors: AP Del Carro; E Rosset, A Josson‐Schramme, V Lambert, S Buff
Pages: n/a - n/a
Abstract: An eight‐month‐old female dog presented with ambiguous external genitalia. A thorough clinical examination together with various imaging techniques and a histology examination showed the presence of two testicles linked to both the Mullerian and Wolffian ducts. The discovery of the 78,XX SRY‐negative karyotype led to the diagnosis of incoherence between the chromosomal and gonadal sex, which is typical for a 78,XX testicular disorder of sex development. Our case was unique because the testicles were still located in their normal scrotal position, whereas the literature contains reports of the presence of cryptorchid testicles in this karyotype setting. To our knowledge, this is the first case that describes an SRY‐negative 78,XX testicular disorder of sex development with bilateral scrotal testicles.
- Sexual Precocity of Nellore Bulls that are Offspring of Super Precocious,
Precocious and Normal Cows in Extensive Farming Conditions
- Authors: JB Siqueira; E Oba, RO Pinho, SEF Guimarães, TM Neto, JD Guimarães
Pages: n/a - n/a
Abstract: The objective of this work was to verify that mothers classified as super precocious (M1) and precocious (M2) produce more precocious bulls than females classified as normal (M3). This study included 21 186 animals with an average age of 21.29 ± 1.77 months that underwent a breeding soundness evaluation from 1999 to 2008. Of these animals, 2019, 6059 and 13 108 were offspring of M1, M2 and M3 females, respectively. In the breeding soundness examination, the animals were classified as sound for reproduction, sound under a natural mating regime, unsound for reproduction and discarded. To compare the averages obtained for each category of mother within the individual breeding soundness classes, a chi‐square test with a 5% error probability was used, considering the effects of year and month of birth and farm. For the three classes of mothers (M1, M2 and M3), 67.26, 67.22 and 64.16% of bull calves were considered sound for reproduction and 19.71, 19.46 and 21.90% were considered unsound for reproduction, respectively. There was no difference in the frequency of animals that were sound for reproduction under the natural breeding regime between the three classes of mothers (8.87, 9.31 and 9.19%, respectively). There was a difference between the numbers of precocious and normal females that were discarded, with frequencies of 4.01 and 4.75%, respectively (p
- Cytometric Analysis of Surface Molecules of Leucocytes and Phagocytic
Activity of Granulocytes and Monocytes/Macrophages in Cows with Pyometra
- Authors: P Brodzki; K Kostro, A Brodzki, K Niemczuk, U Lisiecka
Pages: n/a - n/a
Abstract: Pyometra is a serious problem in dairy cow herds, causing large economic losses due to infertility. The development of pyometra depends mainly on the immunological status of the cow. The aim of the study was a comparative evaluation of selected indicators involving non‐specific and specific immunity in cows with pyometra and in cows without inflammation of the uterus. The study was performed in 20 cows, which were divided into two groups: pyometra group and healthy group, each comprising 10 cows, based on the results of cytological and ultrasonographic tests. A flow cytometric analysis was performed for the surface molecules CD4, CD8, CD14, CD21, CD25 and CD4+CD25+ on leucocytes, and the phagocytic activity was determined from granulocytes and monocytes/macrophages in the peripheral blood and uterine washings, respectively. It was demonstrated that the percentage of phagocytic granulocytes and monocytes/macrophages in both the peripheral blood and uterine washings was significantly lower in cows with pyometra compared with the healthy group (p
- Influence of Gonadotrophin‐Induced First Oestrus on Gilt Fertility
- Authors: DM Hidalgo; RM Friendship, L Greiner, R Manjarín, MR Amezcua, JC Domínguez, RN Kirkwood
Pages: n/a - n/a
Abstract: The aim of this study was to determine the association between the oestrous response of pre‐pubertal gilts to gonadotrophin injection or boar exposure and their subsequent farrowing rate and litter size. At 154 days of age, randomly selected pre‐pubertal gilts received an intramuscular injection of 400 IU equine chorionic gonadotrophin plus 200 IU human chorionic gonadotrophin (PG600®; Merck Animal Health; n = 181). From the remaining pool of animals not treated with hormones, the first gilts showing signs of oestrus were selected to act as controls (n = 201). Boar exposure began at 155 days of age for both groups, and gilts were bred at a weight of approximately 130 kg. Comparisons were made between PG600®‐treated gilts exhibiting oestrus or not within 7 days post‐injection (early and late responders, respectively) and control gilts exhibiting oestrus or not within 30 days after beginning of boar exposure (select and non‐select control gilts, respectively). By 162 days, oestrus was detected in 67.5% of PG600®‐treated gilts compared with 5.7% of control gilts (p
- Use of Cholesterol‐Loaded Cyclodextrin in Donkey Semen
Cryopreservation Improves Sperm Viability but Results in Low Fertility in
- Authors: RR Oliveira; DM Rates, G Pugliesi, PG Ker, RP Arruda, EA Moraes, GR Carvalho
Pages: n/a - n/a
Abstract: The use of cholesterol‐loaded cyclodextrin (CLC) on semen cryopreservation has been related with better sperm viability in several species; however, the effect on fertility is not known in donkey semen. Ejaculates (n = 25) from five donkeys were diluted in S‐MEDIUM with 0, 1, 2 or 3 mg of CLC/120 × 106 spermatozoa. Semen was frozen, and thawed samples were evaluated by computer‐assisted sperm analyser system (CASA), supravital test, hyposmotic swelling test and fluorescent dyes to assess the integrity of sperm membranes. Mares (n = 60) were inseminated with frozen‐thawed semen treated with the doses of 0 or 1 mg CLC. Percentages of sperm with progressive motility and with functional plasma membrane were greater (p
- Comparison of Conventional Freezing and Vitrification with
Dimethylformamide and Ethylene Glycol for Cryopreservation of Ovine
- Authors: FC Varago; VS Moutacas, BC Carvalho, RV Serapião, F Vieira, H Chiarini‐Garcia, FZ Brandão, LS Camargo, M Henry, MA Lagares
Pages: n/a - n/a
Abstract: The aim of this work was to evaluate the efficiency of the cryoprotectants dimethylformamide and ethylene glycol for cryopreservation of ovine embryos using vitrification and conventional freezing. The recovered embryos were distributed randomly in three treatment groups: Gr. 1: conventional freezing (n = 44), Gr. 2: vitrification with ethylene glycol (n = 39) and Gr. 3: vitrification with dimethylformamide (n = 38). Quality of fresh embryos in control group as well as of frozen and vitrified embryos was examined by three methodologies: staining with propidium iodide and Hoechst 33258 and evaluation under fluorescent microscopy, evaluation of re‐expansion and hatching rates after culture, and determination of apoptotic index with TUNEL technique. It was established that re‐expansion rate in all treatment groups was similar. In the same time, hatching rates were higher in Gr. 1 (40.5%) and Gr. 2 (35.3%) in comparison with Gr. 3 (15.5%, p
- Altered Expression of Transforming Growth Factor‐Beta Isoforms in
Bovine Cystic Ovarian Disease
- Authors: V Matiller; ML Stangaferro, PU Díaz, HH Ortega, F Rey, E Huber, NR Salvetti
Pages: n/a - n/a
Abstract: Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra‐ovarian factors may contribute to follicular persistence. Transforming growth factor‐beta (TGFB) isoforms are important paracrine and autocrine signalling molecules that regulate ovarian follicle growth and physiology. Considering the importance of these factors in the ovarian physiology, in this study, we examined the expression of TGFB isoforms (TGFB1, TGFB2 and TGFB3) in the ovary of healthy cows and animals with spontaneous and adrenocorticotrophic hormone (ACTH)‐induced COD. In the oestrous‐synchronized control group, the expression of TGFB1 in granulosa and theca cells was higher in spontaneous cysts than in atretic or tertiary follicles. When we compared TGFB2 expression in granulosa cells from atretic or tertiary follicles from the oestrous‐synchronized control group with that in ACTH‐induced or spontaneous follicular cysts, we found a higher expression in the latter. The expression of the TGFB isoforms studied was also altered during folliculogenesis in both the spontaneous and ACTH‐induced COD groups. As it has been previously shown that TGFB influences steroidogenesis, ovarian follicular proliferation and apoptosis, an alteration in its expression may contribute to the pathogenesis of this disease.
- The Expression and Localization of Crb3 in Developmental Stages of the
Mice Embryos and in Different Organs of 1‐week‐old Female Mice
- Authors: Y Yin; J Sheng, R Hu, Y Yang, S Qing
Pages: n/a - n/a
Abstract: Crumbs homolog 3 (Crb3) is a member of the Crumbs family of proteins. This protein may play a role in epithelial cell polarity and is associated with tight junctions at the apical surface of epithelial cells. Alternative transcriptional splice variants that encode different Crb3 isoforms have been characterized. The expression of Crb3 mRNA and protein was observed in the pre‐implantation mouse embryos and different organs of 1‐week‐old mouse, and Crb3 expression was primarily observed in the cytoplasm. Crb3 was expressed in a unique temporal pattern in pre‐implantation embryos. The main characteristic of Crb3 expression was that the positive signals were stronger in the mature oocytes and zygotes than in the 2‐cell, 4‐cell, and 8‐cell stages and the morula, but a similar level of high expression was observed in blastocysts. Therefore, the Crb3 expression signal during the course of development process grew gradually stronger from the 2‐cell stage to blastocyst. In addition, Crb3 protein was widely distributed in each stage of the post‐implantation embryos. Crb3 expression was observed in the inner cell mass, trophoblast cells and endoderm of E4.5d embryos; in the chorion, amnion, trophoblast cells, yolk sac endoderm and embryo ectoderm of E7.5d embryos; in the amnion and limb bud of E8.0d embryos; and in the semicircular canal epithelium, retina, lens vesicle and liver tissue of E13.5d embryos. Crb3 was expressed at different levels in different organs of 1‐week‐old mouse with the strengths in the following order: kidney > small intestine > stomach > uterus > liver > skeletal muscle > cerebellum > brain. The presence of Crb3 in many organs and the regularity of Crb3 distribution in the process of mouse embryonic development indicate that Crb3 protein plays an important role in establishing and maintaining the polarity of mouse embryos.
- Effects of Prolonged in vitro Culture and Cryopreservation on Viability,
DNA Fragmentation, Chromosome Stability and Ultrastructure of Bovine Cells
from Amniotic Fluid and Umbilical Cord
- Authors: ER Cunha; CF Martins, CG Silva, HC Bessler, SN Báo
Pages: n/a - n/a
Abstract: The objective of this work was to study cellular types that did not participated in the gastrulation process, amniotic fluid cells (AFCs) and umbilical cord cells (UCCs), in conditions of long‐term culture and cryopreserved with different solutions. The AFCs and UCCs were used in a comparative study with ear fibroblast cells (EFCs) that were cultured in vitro until 20 cellular passages and cryopreserved in 10% dimethylsulphoxide (DMSO), 5% dimethyl formamide (DMF) and 7% glycerol (Gly) solutions. The cellular viability, ultrastructure, DNA fragmentation and chromosome stability were evaluated to determine the cellular type most resistant. In all cell types, it was possible to evaluate the AFCs until 15 passages and UCCs until 20 passages with different periods of cellular growth to reach the confluence phase. Solutions containing 10% DMSO ensured viability of 90.33 ± 5.58%, 90.56 ± 4.40% and 81.90 ± 3.31%, respectively for EFCs, AFCs and UCCs, being significantly more efficient and with less variation than other cryoprotectant solutions. The AFCs were more sensitive to cryopreservation and presented low viability rate at the passage 20 (17.2 ± 8.87%). There was no change in karyotype and nuclear fragmentation was low in all cellular passages studied. With the scanning electron analysis was possible the characterization of AFCs and UCCs in suspension. The three cellular types of cells presented different shapes and characteristics on the surface. The results demonstrate that bovine AFCs and UCCs can be isolated, cultured in vitro and cryopreserved in 10% DMSO, not causing damage to DNA and chromosomes. The UCCs were more resistant than AFCs in all aspects.
- ‘Soya Milk Tris‐based Phytoextender Reduces Apoptosis in
Cryopreserved Buffalo (Bubalus bubalis) Spermatozoa’
- Authors: R Mohan; SK Atreja
Pages: n/a - n/a
Abstract: The objective of this study was to investigate the effect of newly developed soya milk Tris (SMT)‐based phytoextender as an alternative to egg yolk Tris (EYT) extender used for cryopreservation of buffalo (Bubalus bubalis) spermatozoa on apoptosis. Fresh buffalo semen (control without dilution) was cryopreserved in conventional EYT (20% egg yolk v/v in Tris) and SMT (25% soya milk v/v in Tris) extender and used for the assessment of expression of apoptotic proteins. Proteins extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random were separated using SDS–PAGE followed by immunoblotting against caspase‐8, caspase‐9, caspase‐3, poly(ADP‐ribose)polymerase (PARP), cytochrome c and apoptosis inducing factor (AIF). In addition, fluorescence microscopy was used for the detection of mitochondrial membrane potential (JC‐1 assay) and apoptotic cells (annexin V‐FITC/PI assay). The results obtained clearly indicate the significant (p 0.05) reduction in expression of PARP‐DNA‐binding subunit (24 kDa) was observed in SMT extender. No expression of AIF was found in cryopreserved semen samples. A significant (p 0.05) decrease in late apoptotic cells (AN+/PI+) was observed in SMT extender when compared to EYT extender. The results demonstrated that cryopreservation of buffalo semen in SMT‐based phytoextender can replace the traditional egg yolk extenders as it reduces the expression of apoptotic proteins maintaining high mitochondrial membrane potential and gives better protection to sperms in terms of its non‐animal origin.
- Evaluation of the Breeding Soundness of Male Camels (Camelus dromedarius)
via Clinical Examination, Semen Analysis, Ultrasonography and Testicular
Biopsy: A Summary of 80 Clinical Cases
- Authors: MM Waheed; IM Ghoneim, MM Hassieb, AA Alsumait
Pages: n/a - n/a
Abstract: Male camel infertility is a heterogeneous disorder. A variety of factors may adversely affect sperm production and function and impair fertility. This study was designed to evaluate the sensitivity and specificity of ultrasonography and testicular biopsy in the evaluation of the breeding soundness of male dromedaries compared with results obtained by clinical examination and semen analysis. Eighty‐four male dromedary camels (5–15 years old) were used in this study during the rutting season (November–May). Four sexually mature male camels were used as controls. These animals were apparently healthy and had histories of normal fertility. Eighty infertile male camels were subjected to an algorithmic approach based on information collected during careful examinations of the camels' breeding histories, clinical examinations, testicular evaluations, testicular ultrasonographies, the results of the semen analyses and testicular biopsies to diagnose the camels' infertilities. The differences in the semen parameters between the control and infertile male camels were highly significant (p
- MALDI‐MS Lipid Profiles of Oocytes Recovered by Ovum Pickup from Bos
indicus and 1/2 indicus × taurus with High vs Low Oocyte Yields
- Authors: KC Silva‐Santos; CR Ferreira, GMG Santos, MN Eberlin, LS Siloto, CO Rosa, TN Marcantonio, MM Seneda
Pages: n/a - n/a
Abstract: The aim of the present study was to compare the lipid profile in oocytes of indicus and 1/2 indicus × taurus cows with high and low antral follicle count (AFC)/oocyte yields. After an OPU procedure (D0), antral follicles ≥3 mm were counted by ultrasonography (D4, 19, 34, 49, 64), and cows were assigned to groups with either high AFC (≥30 follicles; indicus, NH group; 1/2 indicus × taurus, AH group) or low AFC (≤15 antral follicles; indicus, NL group; 1/2 indicus × taurus, AL group). The lipid profiles of the oocytes were determined by MALDI‐MS. For GI, GII and GIII oocytes, the indicus samples tend to cluster separately from the 1/2 indicus × taurus samples. The lipid species [PC (P‐38:5) + H]+ and/or [PC (P‐36:2) + Na]+, [PC (38:2) + H]+, [PC (38:5) + Na]+ and [TAG (60:8) + NH4]+ were more abundant in indicus (NH and NL groups) than 1/2 indicus × taurus. The higher lipid content in the indicus oocytes likely reflects differences in the rate of lipid metabolism and may contribute to oocyte competence and embryo development.
- Protein Localization of Epidermal Growth Factor in Sheep Ovaries and
Improvement of Follicle Survival and Antrum Formation In Vitro
- Authors: LP Santos; VRP Barros, AYP Cavalcante, VG Menezes, TJS Macedo, JMS Santos, VR Araújo, MAA Queiroz, MHT Matos
Pages: n/a - n/a
Abstract: The aims of this study were to characterize EGF protein expression in ovine ovaries and to verify the effect of EGF on the in vitro development of isolated pre‐antral follicles. After collection, ovarian tissue was fixed for immunohistochemical analysis. Additional pairs of ovaries were collected, and secondary follicles were cultured for 18 days in α‐MEM+ (control) alone or supplemented with EGF (1, 10 or 50 ng/ml). The immunostaining for EGF was observed in oocytes from pre‐antral and antral follicles, in granulosa cells of primary and secondary follicles, as well as in cumulus and mural cells of antral follicles. After 18 days, the results showed that treatment with 50 ng/ml EGF significantly increased the percentage of morphologically normal follicles compared with the control group (α‐MEM+) and significantly reduced the precocious extrusion of oocytes and increased the percentage of antral follicles compared with the control and 1 ng/ml EGF. All the treatments induced a progressive and significant increase of the follicular diameter throughout the period of culture. However, there were no significant differences in follicular diameter or in the daily growth rate among treatments. In conclusion, this study demonstrated the presence of EGF in ovine ovaries. Moreover, 50 ng/ml EGF increased the percentage of normal follicles and improved antrum formation in isolated ovine follicles after 18 days of in vitro culture.
- Influence of Temperament Score and Handling Facility on Stress,
Reproductive Hormone Concentrations, and Fixed Time AI Pregnancy Rates in
- Authors: R Kasimanickam; S Schroeder, M Assay, V Kasimanickam, DA Moore, JM Gay, WD Whittier
Pages: n/a - n/a
Abstract: The objectives were (i) to evaluate the effect of temperament, determined by modified 2‐point chute exit and gait score, on artificial insemination (AI) pregnancy rates in beef heifers following fixed time AI and (ii) to determine the effect of temperament on cortisol, substance‐P, prolactin and progesterone at initiation of synchronization and at the time of AI. Angus beef heifers (n = 967) at eight locations were included in this study. At the initiation of synchronization (Day 0 = initiation of synchronization), all heifers received a body condition score (BCS), and temperament score (0 = calm; slow exit and walk or 1 = excitable; fast exit or jump or trot or run). Blood samples were collected from a sub‐population of heifers (n = 86) at both synchronization initiation and the time of AI to determine the differences in serum progesterone, cortisol, prolactin and substance‐P concentrations between temperament groups. Heifers were synchronized with 5‐day CO‐Synch+ controlled internal drug release (CIDR) protocol and were inseminated at 56 h after CIDR removal. Heifers were examined for pregnancy by ultrasound 70 days after AI to determine AI pregnancy. Controlling for synchronization treatment (p = 0.03), facility design (p = 0.05), and cattle handling facility design by temperament score interaction (p = 0.02), the AI pregnancy differed between heifers with excitable and calm temperament (51.9% vs 60.3%; p = 0.01). The alley‐way with acute bends and turns, and long straight alley‐way had lower AI pregnancy rate than did the semicircular alley‐way (53.5%, 56.3% and 67.0% respectively; p = 0.05). The serum hormone concentrations differed significantly between different types of cattle handling facility (p
- Efficacy of Low‐Dose Human Chorionic Gonadotropin Therapy in Dogs
with Spermatogenic Dysfunction: A Preliminary Study
- Authors: M Kobayashi; T Hori, E Kawakami
Pages: n/a - n/a
Abstract: Human chorionic gonadotropin (hCG) is a glycoprotein used in the treatment of spermatogenic dysfunction. However, previous studies performed in dogs show that repeated administration of large doses of hCG produces antibodies against hCG. In this study, we examined the efficacy of low‐dose injections of hCG in four male dogs with spermatogenic dysfunction and low plasma testosterone (T) levels. We administered 100 IU hCG per animal, five times at 3‐day intervals, and evaluated the changes in semen quality and plasma T levels. The total number of sperm in ejaculate, the percentage of progressively motile sperm and the plasma T levels had increased by 3–5 weeks after the first injection of hCG in three of the four dogs, but were unchanged in the fourth dog. These findings indicate that temporary improvement of the semen quality of dogs with spermatogenic dysfunction and low plasma T levels is possible after five low‐dose injections of hCG.
- Effect of Three pFSH Doses on Superovulation and Embryo Quality in Goats
During Two Breeding Seasons in North-eastern Mexico
- Authors: F Sánchez-Dávila; RA Ledezma-Torres, G Padilla-Rivas, AS Bosque-González, A González Gómez, H Bernal-Barragán
Pages: e40 - e43
Abstract: The aim of this research was to evaluate the effectiveness of three pFSH doses (80 mg; 145 mg and 215 mg) on ovarian response and on quantity and quality of transferable embryos of goats during the breeding and the non-breeding seasons. Ovary structures were exposed (laparatomy under general anaesthesia) and numbers of follicles and corpora lutea were registered. Surgical embryo flushing was conducted to count and classify embryos. There were more follicles (3.4 ± 1.1) in does administered 80 mg of pFSH (p
- Impact of Probing the Reproductive Tract During Early Pregnancy on
Fertility of Beef Cows
- Authors: G Pugliesi; SC Scolari, FS Mesquita, M Maturana Filho, ER Araújo, D Cardoso, JN Sales, I Martin, M Sá Filho, CM Bertan, M Binelli
Abstract: This short communication reports the impact of endometrial biopsies, uterine flushings and follicular fluid aspiration procedures at day 6 post artificial insemination (AI) on pregnancy rates. In Experiment 1, cows were timed AI (TAI) and assigned to the following treatment groups: control (n = 37), uterine flushing (n = 35) and endometrial biopsy (n = 38). On day 30 post AI, pregnancy rates were 40.5%, 33% and 28.5%, respectively (p > 0.1). Pregnancy rate on day 60 was lower (p 0.1). In Experiment 3, cows were, 6 days after TAI, randomly assigned to the following treatments: control (n = 84) or aspiration of the largest follicle (n = 73). Pregnancy rates on day 30 post AI were 63.5% for the control group and 53% for the aspirated group (p > 0.1). In conclusion, uterine flushing and endometrial biopsy negatively affect pregnancy rates, but neither procedure can be considered to be incompatible with pregnancy maintenance. Follicular aspiration during pregnancy does not interact with pregnancy success. The amount and quality of samples obtained are compatible with the use of cellular and molecular analysis of uterine variables from cows that failed or succeeded on maintaining pregnancy.
- Does Coxiella burnetii Affect Reproduction in Cattle? A Clinical
- Authors: I Garcia-Ispierto; J Tutusaus, F López-Gatius
Pages: 529 - 535
Abstract: Q fever is a zoonosis produced by Coxiella burnetii, a bacterium that is widely distributed worldwide. Domestic ruminants are the most important source of C. burnetii for human infection. In sheep and goats, abortion is the main clinical consequence of infection, yet the symptoms described in cattle have so far been inconsistent. Q fever has been also scarcely reported in cattle, most likely because of its difficult diagnosis at the farm level and because of the many existing responsible C. burnetii strains. In this report, the effects of C. burnetii infection or Q fever disease on the reproductive behaviour of dairy cattle are reviewed, with special emphasis placed on the scarcity of data available and possible control actions discussed.
- Are Pharmacological Interventions Between Conception and Birth Effective
in Improving Reproductive Outcomes in North American Swine'
- Authors: JM Wessels; K Khalaj, RT Kridli, AK Edwards, M Bidarimath, C Tayade
First page: 536
Abstract: The objective of this review is to evaluate the effectiveness of using pharmacological compounds on reproductive outcomes, particularly litter size, in North American swine. While the opportunity to improve reproduction in North American pigs exists, numerous hurdles need to be overcome in order to achieve measureable results. In the swine industry, the majority of piglet losses are incurred during pregnancy and around farrowing. Over the last 20 years, a reduction in losses has been achieved through genetic selection and nutritional management; however, these topics are the focus of other reviews. This review will evaluate attempts to improve litter size by reducing losses at various stages of the reproductive process, from the time of conception to the time of farrowing, using pharmacological compounds. Generally, these compounds are used to either alter physiological processes related to fertilization, embryonic attachment or uterine capacity, etc., or to facilitate management aspects of the breeding females such as inducing parturition. Although some of the pharmacological agents reviewed here show some positive effects on improving reproductive parameters, the inconsistent results and associated risks usually outweigh the benefits gained. Thus, at the present time, the use of pharmacological agents to enhance reproduction in North American swine may only be recommended for herds with low fertility and presents an avenue of research that could be further explored.
- Apoptosis-Like Events and In Vitro Fertilization Capacity of Sex-Sorted
- Authors: X-M Zhao; J-J Ren, S-J Zhao, L-S Cui, H-S Hao, H-Y Wang, W-H Du, T Qin, Y Liu, D Wang, H-B Zhu
First page: 543
Abstract: This study utilized three staining assays (Annexin V, mitochondrial membrane potential (JC-1) and TUNEL) for flow cytometric analysis of apoptosis in sex-sorted sperm from four different bulls (A, B, C and D). Correlations between sperm quality and IVF efficiency were then assessed to determine which assay provided the best prediction of IVF efficiency. The results of the Annexin V assays, as well as measures of viable sperm, early apoptosis, necrotic sperm and mitochondrial membrane potential (∆ψm) showed that the sex-sorted sperm collected from bull A significantly differed from those of the other three bulls (p
- The Endometrium of Cycling Cows Contains Populations of Putative
Mesenchymal Progenitor Cells
- Authors: J Cabezas; E Lara, P Pacha, D Rojas, D Veraguas, F Saravia, L Rodríguez-Alvarez, FO Castro
Pages: 550 - 559
Abstract: Endometrial stem cells have been identified in humans, mice and pigs. This study was designed to determine whether the uterine endometrium of cycling cows contains such cells, to identify markers of stemness and ultimately to isolate putative stem/progenitor cell and evaluate their capability to differentiate into mesodermal derivatives. Uteri from healthy cows in the early (days 1–5) and late luteal phases (days 13–18) of the oestrous cycle were collected. Total RNA and proteins were isolated and searched for gene markers of embryonic (OCT4, NANOG, SOX2) and mesenchymal (CD44, STAT3, CD-117) stem cells and for protein markers (Oct4, Sox2, Cd44) in Western blots or immunostaining of paraffin-embedded tissue. Primary cell cultures were isolated; characterized in terms of morphology, colony formation and gene/protein expression; and induced osteogenic and chondrogenic differentiation. We identified expression of embryonic (OCT4 and SOX2, but not NANOG) and mesenchymal (STAT3, CD44 and c-KIT) gene markers in the endometrium of cycling cows and the encoded proteins (Oct4, Sox2 and Cd44) in both stages of the oestrous cycle. Derived cell lines displayed essentially the same gene expression pattern; however, at the protein level, Oct4 was not detected. No clear influence of the stage of the oestrous cycle was found. Cell lines from late luteal phase displayed osteogenic and chondrogenic differentiation potential upon chemical stimulation. In this research, we demonstrated the presence of mesenchymal progenitor cell populations of apparently mesenchymal origin in the endometrium of cycling cows, in both the early and late phases of the oestrous cycle. The cells isolated from the late luteal phase were more acquiescent to differentiate into mesodermal derivatives than cells in the early luteal phase. Our findings might have implications for the understanding of uterine stem cell biology in cows and other farm animal species.
- Kinematics and Subpopulations’ Structure Definition of Blue Fox
(Alopex lagopus) Sperm Motility using the ISAS® V1 CASA System
- Authors: C Soler; A García, J Contell, J Segervall, M Sancho
First page: 560
Abstract: Over recent years, technological advances have brought innovation in assisted reproduction to the agriculture. Fox species are of great economical interest in some countries, but their semen characteristics have not been studied enough. To advance the knowledge of function of fox spermatozoa, five samples were obtained by masturbation, in the breeding season. Kinetic analysis was performed using ISAS® v1 system. Usual kinematic parameters (VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were considered. To establish the standardization for the analysis of samples, the minimum number of cells to analyse and the minimum number of fields to capture were defined. In the second step, the presence of subpopulations in blue fox semen was analysed. The minimum number of cells to test was 30, because kinematic parameters remained constant along the groups of analysis. Also, the effectiveness of ISAS® D4C20 counting chamber was studied, showing that the first five squares presented equivalent results, while in the squares six and seven, the kinematic parameters showed a reduction in all of them, but not in the concentration or motility percentage. Kinematic variables were grouped into two principal components (PC). A linear movement characterized PC1, while PC2 showed an oscillatory movement. Three subpopulations were found, varying in structure among different animals.
- The Effect of the Intracervical Application of Follicle-Stimulating
Hormone or Luteinizing Hormone on the Pattern of Expression of
Gonadotrophin Receptors in the Cervix of Non-Pregnant Ewes
- Authors: S Leethongdee; M Khalid, RJ Scaramuzzi
Pages: 568 - 575
Abstract: During the periovulatory period, the cervix relaxes in response to changes in circulating concentrations of reproductive hormones. The present study investigated the role of gonadotrophins in cervical function by examining the expression of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) and their mRNAs following intracervical treatment with either FSH or LH. Eighteen ewes were assigned to four groups, and they were then treated with progestagen sponges and PMSG to synchronize their oestrous cycles. Intracervical treatments were given 24 h after sponge removal as follows: Group 1: FSH 2 mg; Group 2: LH 2 mg; Group 3: Vehicle and Group 4: Control. Cervices were collected 54 h after sponge removal and then divided into three regions. The expression of FSHR and LHR was determined by immunohistochemistry and FSHR mRNA and LH mRNA by in situ hybridization. The expression of LHR, FSHR and their respective mRNAs was compared in six tissue layers (luminal epithelium, subepithelial stroma, circular, longitudinal and transverse muscle and serosa) and in three cervical regions (vaginal, mid and uterine). The results showed that FSH increased transcription of the FSHR gene and the levels of its receptor, but only in subepithelial stroma of the cervix. FSH also increased the levels of LHR in the cervix, but only in the muscle layers. LH had no effect on the levels of FSHR despite the fact that it did increase the level of transcription of the FSHR gene and LH also increased the levels of its own receptor in the cervix, but only in the muscle layers, and this action was independent of increased levels of transcription of the LHR gene. These findings suggest multiple levels of regulation of cervical LH and FSH receptors and that the gonadotrophins may have a role in relaxation of the cervix during oestrus by regulating their own receptors.
- The Influence of Morphology, Follicle Size and Bcl-2 and Bax Transcripts
on the Developmental Competence of Bovine Oocytes
- Authors: THC De Bem; PR Adona, FF Bressan, LG Mesquita, MR Chiaratti, FV Meirelles, CLV Leal
First page: 576
Abstract: This study analysed two non-invasive oocyte selection methods in relation to in vitro embryo development capacity and expression of apoptosis-related genes. Selection was based on morphological quality of oocytes or follicle diameter. Oocytes were classified as grade I (GI ≥3 layers compact cumulus cells and homogeneous cytoplasm; grade II (GII ≤3 layers compact cells and homogeneous cytoplasm;, and grade III (GIII ≥3 layers, but cells with slight expansion and slightly granulated cytoplasm). Blastocyst development was lower for GII (28.5%) than for GIII (47.7%, p 0.05). Relative expression of Bcl-2 gene was lower in the GI (1.0, p 0.05). There was no difference (p > 0.05) between GI (1.0), GII (0.92) and GIII (0.93) regarding the Bax transcript. However, the Bax and Bcl-2 transcript ratios in GII (Bax; 0.92 and Bcl-2; 1.8) and GIII (Bax; 0.93 and Bcl-2; 2.2) were different (p 0.05). Regarding Bax and Bcl-2 expression, the oocytes were similar (p > 0.05) for 1–3 mm (Bax; 1.0 and Bcl-2; 1.0), 3–6 mm (Bax; 1.0 and Bcl-2; 0.93) and 6–9 mm (Bax; 0.92 and Bcl-2; 0.91). In conclusion, oocyte selection based on morphological appearance does not guarantee the success of embryonic development. Additionally, the absence of apoptosis is not necessarily a benefit for the development of oocytes. Bovine COCs with initial signs of atresia may be used for the in vitro production of embryos, and COCs taken from follicles>3 mm in diameter are better suited to in vitro embryo development.
- Genetic Parameters and Breeding Values for Semen Characteristics in
- Authors: D Labitzke; H Sieme, G Martinsson, O Distl
First page: 584
Abstract: The objectives of this study were to show whether semen traits of 30 Hanoverian stallions regularly used in AI may be useful for breeding purposes. Semen characteristics were studied using 15 149 ejaculates from 30 Hanoverian stallions of the State Stud Celle of Lower Saxony. Semen samples were collected between 2005 and 2009. Traits analysed were gel-free volume, sperm concentration, total and motile sperm number and progressive motility. A linear multivariate animal model was employed to estimate heritabilities and permanent environmental variances for stallions. The same model was used to predict breeding values for all traits simultaneously. Heritabilities were high for gel-free volume (h2 = 0.43) and moderate for total number of sperm (h2 = 0.29) and progressive motility (h2 = 0.20). Gel-free volume, sperm concentration and total number of sperm were genetically negatively correlated with progressive motility. The effect of the permanent environment for stallions accounted for 9–55% of the trait variance. The total variance among stallions explained 37–69% of the trait variance. The average reliabilities of the breeding values were 0.43–0.76 for the 30 Hanoverian stallions. In conclusion, the study could demonstrate large effects of stallions, routinely employed in a breeding programme, on semen characteristics analysed here. We could demonstrate that estimated breeding values (EBV) with sufficient high reliabilities can be predicted using data from these stallions and these EBV are useful in horse breeding programmes to achieve genetic improvement in semen quality.
- Induction of Ram Bone Marrow Mesenchymal Stem Cells into Germ Cell Lineage
using Transforming Growth Factor-β Superfamily Growth Factors
- Authors: M Ghasemzadeh-Hasankolaei; MA Sedighi-Gilani, MB Eslaminejad
Pages: 588 - 598
Abstract: Several studies have proposed that in vitro generation of germ cells (GCs) from stem cells can be considered a future option for infertility treatment. Mesenchymal stem cells (MSCs) have the capability to differentiate into male GCs with the use of inducers such as retinoic acid. Transforming growth factor-beta 1 (TGFb1) has been shown to play important roles in male fertility and spermatogenesis. Bone morphogenic protein 4 (BMP4) and BMP8b are also involved in the derivation of primordial GCs (PGCs) from epiblast cells. Therefore, this study aims to determine whether TGFb1, BMP4 and BMP8b can initiate transdifferentiation of MSCs into GCs in vitro and to determine the type of changes that occur in the expression of GC-specific markers. In this study, we have divided passage-3 ram bone marrow (BM)-MSCs into three main groups (BMP4, BMP8b and TGFb1) which were separately treated with 10 ng/ml TGFb1, 100 ng/ml BMP4 and 100 ng/ml BMP8b for a period of 21 days. We have evaluated the ability of these groups to differentiate into GCs by assessing expressions of GC-specific markers with reverse transcription PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), immunocytochemistry, morphological changes and alkaline phosphatase (ALP) activity. Our results showed that BMP4 and BMP8b induced PGCs properties in some BM-MSCs and TGFb1 formed spermatogonial stem cells (SSCs) and spermatogonia-like cells in BM-MSCs culture. The important results of this study provide the basis for additional studies to determine the exact mechanism of GCs differentiation and possibly solve the problem of infertility.
- Improvement in In Vitro Fertilization Rate, Decrease in Reactive Oxygen
Species and Spermatozoa Death Incidence in Rams by Dietary Fish Oil
- Authors: A Matini Behzad; B Ebrahimi, AR Alizadeh, V Esmaeili, A Dalman, L Rashki, AH Shahverdi
Pages: 599 - 605
Abstract: Our aim was to evaluate the effects of fish oil feeding on sperm classical parameters, level of reactive oxygen spices (ROS), spermatozoa death incidence and in vitro fertilization (IVF) rate in rams. We randomly assigned nine rams, into two experimental groups (isoenergetic and isonitrogenous rations with constant level of vitamin E supplement): control (CTR; n = 5) and fish oil (FO; n = 4, 35 g/day/ram). Diets were fed for 70 days during the physiological breeding season. After a 21-day dietary adaptation period, semen was collected weekly from each ram by an artificial vagina. Sperm classical parameters were determined by the computer-assisted sperm analyzer system (CASA), and it was prepared for IVF process by swim-up technique. These evaluations were performed during the first and last weeks of sampling. Intracellular ROS level and spermatozoa death incidence were detected by flow cytometry on a weekly basis after adaptation. Data were analysed with SPSS 15. The volume, concentration (3.6 and 2.7 × 109/ml) and sperm progressive motility (60 and 48%) were significantly improved in the FO group compared with the CTR (p
- Coexistence of Tuberculosis and Mammary Carcinoma in a Goat
- Authors: H Quintas; N Alegria, A Mendonça, A Botelho, A Alves, I Pires
First page: 606
Abstract: Synchronic occurrence of tuberculosis mastitis and mammary cancer is rare in humans and, to the best of our knowledge, not reported in domestic animals. Here, we present a case of a female adult goat of Serrana breed with simultaneous occurrence of a granulomatous mastitis, due to Mycobacterium caprae, and a mammary carcinoma. Both pathological conditions are rare in goats and should be included in differential diagnosis of mammary lesions.
- The Effects of Isoflavones on Androgens and Glucocorticoids During Puberty
on Male Wistar Rats
- Authors: S Caceres; G Silvan, L Martinez-Fernandez, MJ Illera, P Millan, B Monsalve, L Peña, JC Illera
First page: 611
Abstract: Isoflavones are the most common phytoestrogens found in human diets. However, it is still not clear whether isoflavones have effects on the reproductive and the endocrine systems under normal dietary intake and overdose. The aim of this study was to determine how the most important isoflavones, genistein and daidzein, affect androgen and glucorticoid levels on male prepuberal rats. A hundred and seventy-five 30-day-old male Wistar rats were dosed orally by stomach tube every day for 35 days, with saline solution, low and high doses of genistein, daidzein and a mixture of both. Serum samples were analysed by an enzyme immunoassay for hormone determinations. In control group, there was a peak of testosterone (T) and dihydrotestosterone levels associated to the onset of puberty, at the third week. However, in low-dose groups, the same peak was found at the fourth week (p
- Pregnancy-Associated Glycoprotein (PAG) Profile of Holstein–Friesian
Cows as Compared to Dual-Purpose and Beef Cows
- Authors: M Shahin; M Friedrich, M Gauly, W Holtz
First page: 618
Abstract: Pregnancy-associated glycoproteins (PAGs) are produced by mono- and binucleate trophoblast cells in the ruminant placenta. PAG appears in maternal blood and, from approximately 4 weeks after fertilization onward, may serve as a reliable means of diagnosing pregnancy. A range of factors are said to affect plasma PAG concentrations, such as number and sex of foetus, mass of calf and placenta, level of milk production and genetic constitution. In this study, PAG pregnancy profiles of a dual-purpose (Simmental) and two beef breeds (Uckermark and Aubrac) are compared with the profile of the specialized dairy breed Holstein–Friesian. Holstein–Friesian cows were sampled weekly; the levels of the other breeds were presented at 3-week intervals. The overall significant breed difference (p = 0.013) was founded on deviations during the initial 3 weeks of pregnancy and from 23 weeks onward. During the period critical for the detection of pregnancy, between four and 22 weeks, agreement between PAG levels of various breeds was close (p > 0.05). No significant effect of body mass of cow or calf (relative to mass of dam) was detected. These findings imply that the PAG pregnancy test may be executed uniformly irrespective of breed or type of cow, affirming the suitability of the test as a valuable asset for the cattle industry.
- Evaluation of Oestrous Detection in Dairy Cattle Comparing an Automated
Activity Monitoring System to Visual Observation
- Authors: I Michaelis; O Burfeind, W Heuwieser
First page: 621
Abstract: The objective of the study was to evaluate an automated activity monitoring (AAM) system for oestrous detection in dairy cows. Specifically, we set out to determine the oestrous detection efficiency and accuracy of the AAM system and to compare reproductive performance of cows detected either by AAM, by visual observation (VO) or by at least 1 of the 2 methods (EOM). A total of 1004 potential cow-periods from 348 cows were analysed. Oestrous detection rates (EDR) were calculated for 21 day (VWP+21), 42 day (VWP+42), and 63 day (VWP+63) after voluntary waiting period (VWP). Oestrous detection rate did not differ between AAM (42.1%) and VO (37.3%) during VWP+21 (p > 0.05) but was significantly higher in EOM (56.3%, p
- Effect of Exposure to Heatwave During Blastocyst Formation on Reproductive
Performance of Female Rabbits
- Authors: F Marco-Jiménez; C Naturil-Alfonso, DS Peñaranda, E Jiménez-Trigos, FJ García-Diego, JS Vicente
First page: 629
Abstract: We examined the effect of female exposure to heatwave during blastocyst formation on their reproductive performance and its effect on transcriptome in blastocyst and endometrial tissue. In this study, a total of 72 rabbit does were artificially inseminated and divided into two environmental groups 2 days later: does under conventional conditions (maintained between 14–22°C, n = 29) and does heat stressed in a climatic chamber (maintained between 32–37°C, n = 43). The heat-stressed group were kept under these conditions for 3 days and returned to conventional conditions thereafter. Five days post-insemination, 48 does were slaughtered to collect blastocyst and endometrium samples. mRNA transcripts from OCT-4, VEGF, erbB3, Ifn-ɣ, HSP70 and HSP90 were analysed by qRT-PCR. At day 12 of gestation, 24 females were examined by laparoscopy to evaluate implanted embryos and at birth the total kits born and individual weights were recorded. Results revealed no gene expression changes in blastocyst and endometrial tissue under heatwave exposure. Moreover, our results demonstrated that rabbit embryos developed from 8–16 cells to blastocyst during a heatwave did not affect implantation rates, total number of kits born and foetal losses. In summary, these results demonstrate that heatwave period is not a critical point in the reproductive performance of rabbits during blastocyst formation.
- ATP Content, Oxidative Stress and Motility of Beluga (Huso huso) Semen:
Effect of Short-Term Storage
- Authors: MS Aramli
First page: 636
Abstract: An effective technique for short-term storage of semen is essential when processing multiple sperm samples and when semen must be transported from collection sites to hatcheries for the fertilization of ova or to laboratories for cryopreservation. In this experiment, beluga (Huso huso) sperm were used to evaluate the effects of short-term storage on several quality parameters (i.e. motility, adenosine triphosphate (ATP) content and oxidative stress indices). Sperm cells exhibited> 50% motility during 3 days of storage with an average total duration of sperm motility varying from 13.33 ± 5.77 to 278.33 ± 25.65 s, and no motile spermatozoa were recorded after 9 days of storage. The levels of oxidative stress indices (thiobarbituric acid reactive substances and carbonyl derivatives of proteins) and antioxidant activity (superoxide dismutase) increased significantly after 3 days of storage. The ATP content also decreased significantly after 2 days of storage. The results of this study can be used to develop effective reproduction management and cryopreservation protocols for this endangered fish.
- Measurement of C-reactive protein and Prostaglandin F2α Metabolite
Concentrations in Differentiation of Canine Pyometra and Cystic
- Authors: SO Enginler; A Ateş, B Diren Sığırcı, BH Sontaş, K Sönmez, E Karaçam, H Ekici, G Evkuran Dal, A Gürel
First page: 641
Abstract: Canine pyometra is a dioestrus period disease in which systemic inflammatory response syndrome (SIRS) is a common outcome due to the response of the body to the bacterial infection. The purpose of this study was i) to differentiate canine pyometra and cystic endometrial hyperplasia (CEH)/mucometra by measuring serum C-reactive protein (CRP) and prostaglandin F2α metabolite (PGFM) concentrations in blood and ii) to compare serum concentrations of CRP and PGFM in bitches with a pathological uterus (pyometra or CEH/mucometra) to concentrations in bitches with a healthy uterus. Mean CRP concentrations were found significantly higher (p
- Systemic Immunosuppression by Methylprednisolone and Pregnancy Rates in
Goats Undergoing the Transfer of Cloned Embryos
- Authors: C Feltrin; CA Cooper, N Mohamad-Fauzi, VHV Rodrigues, LH Aguiar, S Gaudencio-Neto, LT Martins, CEM Calderón, AS Morais, IS Carneiro, TM Almeida, ING Silva, JL Rodrigues, EA Maga, JD Murray, AB Libório, LR Bertolini, M Bertolini
First page: 648
Abstract: The presence of the zona pellucida has been perceived as a requirement for the oviductal transfer of cloned embryos at early stages of development while protecting the embryo from an immune system response. We hypothesized that steroid hormone therapy could reduce a potential cellular immune response after the transfer of zona-free cloned embryos into the oviduct of recipient female goats. In Experiment 1, seven does were used to study the systemic immunosuppressant effect of the methylprednisolone administration (for 3 days) on blood cell counts. Whole blood was collected prior to treatment with methyprednisolone and then on Days 1, 2, 3, 4, 7, 14, 21 and 28 after the first dose of methylprednisolone for the analysis of haematological parameters. Methylprednisolone treatment significantly reduced circulating white blood cells and neutrophils in comparison with pre-treatment levels, demonstrating a systemic immunosuppressant effect. In Experiment 2, a group of 58 does were used as recipient females to study the effect of administration of methylprednisolone for 3 days on the establishment of pregnancies after the transfer of zona-free cloned embryos into the oviducts. No effects on pregnancy rates on Day 30 were observed regarding the distinct treatment groups (control vs. methylprednisolone), the source of oocytes (in vivo- vs in vitro-matured) or the presence or absence of the zona pellucida in embryos. In summary, methylprednisolone was effective at inducing a systemic immunosuppressed state in goats, but the treatment prior to embryo transfer did not affect pregnancy rates. Moreover, pregnancy rates were similar between zona-free and zona-intact goat cloned embryos.
- Caspase Activation, Hydrogen Peroxide Production and Akt Dephosphorylation
Occur During Stallion Sperm Senescence
- Authors: JM Gallardo Bolaños; C Balao da Silva, P Martín Muñoz, M Plaza Dávila, J Ezquerra, IM Aparicio, JA Tapia, C Ortega Ferrusola, FJ Peña
First page: 657
Abstract: To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.
- Ghrelin Accelerates In Vitro Maturation of Bovine Oocytes
- Authors: E Dovolou; IE Messinis, E Periquesta, K Dafopoulos, A Gutierrez-Adan, GS Amiridis
First page: 665
Abstract: Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap-frozen cumulus cells, oocytes and day-7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelin-treated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin-treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7 ± 2.4%) compared with Ghr24 (17.5 ± 2.4%). Differences were detected in various genes’ expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused over-maturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process.
- Husbandry Factors and the Resumption of Luteal Activity in Open and
Zero-Grazed Dairy Cows in Urban and Peri-Urban Kampala, Uganda
- Authors: BM Kanyima; R Båge, DO Owiny, T Ntallaris, J Lindahl, U Magnusson, MG Nassuna-Musoke
First page: 673
Abstract: The study investigated the influence of selected husbandry factors on interval to resumption of post-partum cyclicity among dairy cows in urban and peri-urban Kampala. A prospective study of 85 day post-partum period of 59 dairy cows in open (n = 38) and zero grazing (n = 21) systems was conducted on 24 farms. Cows of parity 1–6 were recruited starting 15–30 days post-partum. Progesterone (P4) content in milk taken at 10–12 day intervals was analysed using ELISA. The cow P4 profiles were classified into ‘normal’ ( 56 days), ‘ceased’ or ‘prolonged’ (if started
- An Immunohistochemical Observations on the Oviduct of the Goat
- Authors: E Marettová; M Maretta
First page: 679
Abstract: The oviduct has an important role in regulating transport of gametes and fertilization. The main role in these functions has a smooth muscle cells and ciliated epithelium lining the oviduct. All functions are under the influence of hormonal and nervous system. The objective of this study was immunohistochemically to examine the following structures: lining epithelium, smooth muscle cells, elastic fibres and nerve fibres. For this purpose, the following antibodies were used: cytokeratin 18, S-100 protein, acetylated α-tubulin, smooth muscle actin, desmin and elastin. Ciliary and secretory cells of the lining epithelium were positive for cytokeratin 18 and S-100 protein. Cilia and the basal body-associated structures of ciliary cells were positive to acetylated α-tubulin. Smooth muscle cells (SMC) in mucosa and of the muscular layer were positive for α-smooth muscle actin (SMA) and desmin. High density of nerve fibres positively reacted to acetylated α-tubulin and S100 protein was present in the mucosa, muscular layer and serosa. Elastic fibres positive for elastin form a dense network at the base of the mucosal folds and in the muscle layer. A dense network of these fibres is accompanying the blood vessels. It is supposed that together with smooth muscle cells they are involved in the transport of ovum and in blood flow regulation.
- Forced Collapse of the Blastocoel Cavity Improves Developmental Potential
in Cryopreserved Bovine Blastocysts by Slow-Rate Freezing and
- Authors: S-H Min; J-W Kim, Y-H Lee, S-Y Park, P-S Jeong, J-Y Yeon, H Park, K-T Chang, D-B Koo
First page: 684
Abstract: This study was conducted to evaluate the effectiveness of forced collapse of the blastocoel before slow-rate freezing and vitrification of bovine blastocysts. Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production using the embryo transfer technique. However, the low efficiency of frozen–thawed embryos survival and further development is a crucial problem. In this study, bovine in vitro and in vivo blastocysts were slow-rate frozen and vitrified after forced blastocoele collapse (FBC) of the blastocyst cavity by puncturing the blastocoele with a pulled Pasteur pipet. Differences in the developmental potential of frozen–thawed blastocysts derived from FBC and non-FBC groups were found in both slow-rate freezing and vitrification. Furthermore, we found that the total cell number of blastocysts in FBC groups was increased and the index of apoptosis in FBC groups was decreased. Consistent with these results, real-time RT-PCR analysis data showed that expression of the anti-apoptotic Bcl-XL gene was significantly increased by FBC groups, whereas expression of the pro-apoptotic Bax gene was significantly decreased by FBC groups. Our results also showed that pregnancy outcomes in both slow-rate frozen and vitrified bovine in vivo blastocysts could be improved by reducing the fluid content after FBC of the blastocyst cavity. Therefore, we suggest that FBC of the blastocyst cavity with a pulled Pasteur pipet is an effective pre-treatment technique for both slow-rate freezing and vitrification of bovine blastocysts.
- Evaluation of Cheetah and Leopard Spermatozoa Developmental Capability
after Interspecific ICSI with Domestic Cat Oocytes
- Authors: LN Moro; AJ Sestelo, DF Salamone
Pages: 693 - 700
Abstract: The ICSI procedure is potentially of great value for felids, and it has not been extensively studied in these species. The objectives of this work were to determine the best conditions for ICSI in the domestic cat (DC) to generate interspecific embryos by injecting cheetah (Ch) and leopard (Leo) spermatozoa. Firstly, DC oocytes were matured with insulin–transferrin–selenium (ITS) or without it (MM) and cultured using atmospheric (21%) or low (5%) oxygen tension after ICSI. The group ITS-5%O2 showed the highest blastocyst rate (p
- Quantity of IL-2, IL-4, IL-10, INF-γ, TNF-α and KC-Like
Cytokines in Serum of Bitches With Pyometra in Different Stages of
Oestrous Cycle and Pregnancy
- Authors: GS Maciel; RR Uscategui, VT Almeida, MEF Oliveira, MAR Feliciano, WRR Vicente
Pages: 701 - 704
Abstract: The occurrence of the pyometra is most common in the first half of the dioestrus when there is decreased cellular immunity associated with increased serum concentration of progesterone in females. The aim of this study was to determine the immunological profile of bitches with pyometra, studying serum levels of IL-2, IL-4, IL-10, IFN-γ, KC-like and TNF-α and comparing them with those of healthy bitches in anoestrus, dioestrus and pregnant. Forty females were divided into four experimental groups: group 1 (G1): with pyometra (n = 10); group 2 (G2): bitches in the second week of gestation (n = 10); group 3 (G3): in anoestrus (n = 10); and group 4 (G4): in dioestrus (n = 10). The serum levels for IL-2, KC-like, INF-γ and TNF-α were similar for all experimental groups. The values obtained for IL-10 were found increased (p