- Reliable collection of Caspian brown trout (Salmo trutta caspius) sperm
using a catheter
- Authors: MS Aramli; K Golshahi, A Banan, E Sotoudeh
Abstract: The traditional stripping procedure for collecting fish semen is associated with the risk of urine contamination, which may significantly affect semen quality and quantity. The use of a catheter as an alternative method for semen collection may overcome this problem. Therefore, this study compared Caspian brown trout (Salmo trutta caspius) semen parameters (i.e. sperm density, seminal plasma osmolality, motility parameters of spermatozoa analysed using computer‐assisted sperm analysis and fertility) between the traditional stripping method and the use of a catheter. All parameter values of the semen collected with a catheter were significantly higher (p
- Effects of low‐density lipoproteins as additive on quality
parameters and oxidative stress following cryopreservation of mithun (Bos
- Authors: P Perumal; SK Srivastava, SK Ghosh, KK Baruah, S Bag, JS Rajoria, K Kumar, C Rajkhowa, M Pande, N Srivastava
Abstract: Artificial breeding of mithun poses several challenges including lack of standard protocol for cryopreservation of spermatozoa. This is further complicated by harmful effects of hen's egg yolk (EY) as additive in extender. Purified low‐density lipoproteins (LDL) extracted from EY have been shown as beneficial over EY extender for long‐term semen storage in several species. This investigation explored use of LDL versus EY on semen quality and oxidative stress following freezing–thawing of spermatozoa. A total of 25 of 50 ejaculates based on biophysical parameters were selected for the experiment. After diluting with the Tris‐citrate‐glycerol (TCG) extender, each sample was split into three equal aliquots: Group I, control, EY; Group II and Group III contained 8% and 10% purified LDL, respectively. Frozen–thawed samples were evaluated for motility parameters (progressive, and in the bovine cervical mucus penetration test [BCMPT]), viability, sperm and nuclear abnormality, acrosome integrity, and enzymatic (leakage of intracellular contents) and biochemical (oxidative stress) profiles and in vitro fertility (IVF) assay. Study revealed a significant (p
- The use of gelatine in long‐term storage (up to 48 hr) at
5°C preserves the pre‐freezing and post‐thawing quality
of brown bear sperm
- Abstract: Sedimentation of spermatozoa occurs during long‐term liquid storage and this may produce deleterious changes. Our aim was to apply gelatine supplementation during long‐term pre‐freezing storage of bear sperm, applying final dilution and 6% glycerol at room temperature and cool in straws. We tested four models of sperm storage using a 1:1 dilution in TTF‐ULE‐Bear extender (TesT‐fructose‐egg yolk‐glycerol 6%): (i) second 1:1 dilution at room temperature (RT), cooling at 5°C in a tube and final dilution (100 × 106 sperm ml−1) (Standard); (ii) final dilution at RT and cooling in a tube (FD‐Tube); (iii) final dilution at RT and cooling in 0.25 ml plastic straw (FD‐Straw); and (iv) final dilution at RT in extender supplemented with 1.5% gelatine (Gelatine) and cooling in a 0.25 ml plastic straw. A Standard sample was stored at 5°C for 1 hr (Control); the rest of the samples (Standard, FD‐Tube, FD‐Straw, Gelatine) were stored for 24 or 48 hrs before freezing (100 × 106 sperm ml−1, glycerol 6%). The quality of the samples was assessed for motility by CASA, and viability (SYBR‐14/propidium iodide‐PI‐; VIAB), acrosomal status (PNA‐FITC/PI; iACR) and apoptotic status (YO‐PRO‐1/PI; YOPRO‐) by flow cytometry. At pre‐freezing, after 48 hr, Gelatine showed significantly higher viability (for VIAB and YOPRO‐) and progressiveness (PM, LIN and STR). At 48 hr, Gelatine showed similar YOPRO‐, iACR, LIN, STR and ALH respect to Control. At both 24 and 48 h post‐thawing, Gelatine sample had similar scores for YOPRO‐, iACR, LIN, STR, WOB and VIAB (only 24 hr) when compared with Control, and lower for TM, PM, rapidPM, VAP and ALH. No differences were found among others experimental groups with respect to Control. In conclusion, gelatine could be a suitable alternative to preserve the viability and progressive motility of brown bear ejaculates during long‐term pre‐freezing storage at 5°C.
- Effect of estradiol cypionate and GnRH treatment on plasma
estradiol‐17β concentrations, synchronization of ovulation and
on pregnancy rates in suckled beef cows treated with FTAI‐based
- Abstract: Two experiments were conducted to evaluate the effect of different ovulation inducers on E‐17β plasma concentrations, synchronized ovulations and pregnancy rates. In Experiment 1, cows received a progesterone intravaginal device (PID) with 1 g of progesterone (P4) plus 2 mg of estradiol benzoate (EB) (day 0). At PID removal (day 8), cows received 0.150 mg of D‐cloprostenol and were randomly assigned to four treatment groups (n = 10/treatment): Group ECP: 1 mg of estradiol cypionate at PID removal, Group EB: 1 mg of EB 24 hr after PID removal, Group GnRH: 10 μg of GnRH 48 hr after PID removal, Group ECP‐GnRH: 1 mg of ECP at PID removal plus 10 μg of GnRH 48 hr later. Ultrasonographic examinations were performed to detect the dominant follicle and ovulation. GnRH‐treated cows ovulated later (p
- Long‐term Effects of Pyrethrin and Cyfluthrin, a Type II Synthetic
Pyrethroid, Insecticide Applications on Bull Reproductive Parameters
- Authors: JL Stewart; CF Shipley, FA Ireland, VL Jarrell, CL Timlin, DW Shike, TL Felix
Abstract: The objectives of this study were to determine effects of cyfluthrin and pyrethrin spray products, used in combination with cyfluthrin topical and ear tag applications, on bull reproductive parameters over 18 weeks. Angus or Angus x Simmental bulls were randomly assigned to one of three treatment groups: (i) no exposure to pyrethrins/cyfluthrin (CONT; n = 10), (ii) cyfluthrin ear tag and topical applications (ET; n = 10), or (iii) cyfluthrin ear tag, topical, premise spray and pyrethrin fog spray applications (ET+S; n = 8). Bull body weight was measured every 3 week, and body condition score and scrotal circumference were recorded on weeks 0, 9 and 18. Semen and serum were collected every 3 weeks for sperm evaluation and testosterone measurement, respectively. There was a treatment × week interaction (p
- Inhibition of FGF Signalling Pathway Augments the Expression of
Pluripotency and Trophoblast Lineage Marker Genes in Porcine
- Authors: LY Li; MM Li, SF Yang, J Zhang, Z Li, H Zhang, L Zhu, X Zhu, V Verma, Q Liu, D Shi, B Huang
Abstract: The consistent failure to isolate bona fide pluripotent cell lines from livestock indicates that the underlying mechanisms of early lineage specification are poorly defined. Unlike other species, the contrivances of segregation have been comprehensively studied in the mouse. In mouse, FGF/MAPK signalling pathway dictates the segregation of hypoblast (primitive endoderm). However, it is not evident whether this mechanism is also conserved in livestock. Here, in this study, we examined the roles of FGF/MAP kinase signalling pathways in porcine parthenogenetic embryos during the early development. Porcine parthenogenetic embryos were cultured in the medium addition with FGFR inhibitor BGJ398 (10 μm) or DEMOS. Pluripotency‐ and lineage‐related gene expressions in the early porcine embryos were determined. Compared to control, total cell numbers on day 7 were significantly higher (55 ± 5.96 vs 47 ± 1.97, p 0.05). Nonetheless, BGJ398 treatment significantly augmented the expression of pluripotency and trophoblast marker genes (SOX2, OCT4, KLF4 and CDX2), but did not significantly change the expression of NANOG and hypoblast marker gene (GATA4). Furthermore, the addition of FGF signalling agonist (FGF2) during the embryo development significantly decreased the expression of pluripotency and trophoblast marker genes (SOX2, NANOG, KLF4 and CDX2), but no significant effect on the expression of OCT4 and GATA4 was observed. Here, we exhibit that inhibition of FGF signalling could improve the quality of the porcine embryo and escalate the chance to capture pluripotency. Besides, it also promotes the trophoblast development of porcine parthenogenetic embryo. In addition, the data suggested that FGF signalling pathway is dispensable for the segregation of hypoblast and epiblast lineages in porcine embryo during the early development.
- Impact of Food Restriction on the Expression of the Adiponectin System and
Genes in the Hypothalamic–Pituitary–Ovarian Axis of
- Authors: R Wang; M Kuang, H Nie, W Bai, L Sun, F Wang, D Mao, Z Wang
Abstract: Adiponectin, a cytokine secreted typically by adipocytes, has been implicated as a molecular switch between female reproduction and energy balance. The present study was undertaken to investigate the expression of adiponectin system and patterns of genes in the hypothalamic–pituitary–ovary (HPO) axis of food‐restricted pre‐pubertal ewes. Eighteen 2‐month‐old female ewes were assigned to 3 groups after a pre‐feeding ad libitum for 10 days (six in each group): the control group (C), the low‐food‐restricted group (LR) and the high‐food‐restricted group (HR), which were fed with 100%, 70% and 50% of ad libitum food intake, respectively. The hypothalamus, pituitary, ovary and serum were collected after food restriction for 2 months. Results by ELISA showed that food restriction increased serum adiponectin concentrations. Quantitative real‐time PCR showed that the gene transcriptions for adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2) were enhanced in the hypothalamic–pituitary–ovarian (HPO) axis, while KISS‐1/GPR‐54 and gonadotropin‐releasing hormone (GnRH) in the hypothalamus and luteinizing hormone β‐subunit (LHβ) and follicle‐stimulating hormone β‐subunit (FSHβ) in the pituitary were reduced after food restriction. Immunohistochemistry results demonstrated that AdipoR1 localized in the oocytes of follicles in the ovary. These results suggest that the alterations in the expression of adiponectin and its receptors in response to food restriction might negatively influence the HPO axis.
- Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But
Not of Aquaporin 9 in Boar Spermatozoa
- Abstract: Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen‐thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP‐7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid‐piece and principal piece domains in ejaculated spermatozoa. AQP‐9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP‐7, but not that of AQP‐9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP‐9 but relocated AQP‐7 towards the acrosome. AQP‐7, but not AQP‐9, appears as a relevant marker for non‐empirical studies of sperm handling.
- Effects of Growth Hormone on In Situ Culture of Bovine Preantral Follicles
are Dose Dependent
- Abstract: The objective of this study was to evaluate different concentrations of growth hormone (GH) on the development of bovine preantral follicles cultured included in the ovarian tissue (in situ) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross‐breeds defined were cultured in situ for 1 and 7 days in minimal essential medium (α‐MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50 ng/ml). The ovarian fragments non‐cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy (MET). The parameters underwent anova (Tukey′s and Dunnett′s tests) and chi‐square test (χ2). After 7 days of culture, the treatment with 50 ng/ml GH showed no differences with fresh control (p > 0.05) and had greater effectiveness than in the 0, 10 and 25 ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50 ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50 ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50 ng/ml GH. Follicles cultured in 10 ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50 ng/ml GH is the most effective concentration for the development of preantral follicles cultured in situ.
- Associations of Leptin and Pituitary‐Specific Transcription Factor
Genes’ Polymorphisms with Reproduction and Production Traits in
- Authors: MAF Nasr; A Awad, IE El Araby
Abstract: This study aimed to detect the genetic variability in Leptin and Pit‐1 genes using polymerase chain reaction–restriction fragment length polymorphism and DNA sequencing also to explore their possible associations with reproductive and productive traits of Egyptian buffaloes. Regarding Leptin gene, three genotypes (AA, AG and GG) were identified with frequency of 0.54, 0.40 and 0.06, respectively, and the genotypes were distributed according to the Hardy–Weinberg equilibrium. Allele A was comparatively higher than G with frequency of 0.74 and 0.26, respectively. For Pit‐1 gene, the association could not be performed due to the monomorphism (BB). The results showed that AA genotypes were found to be superior in most of production and reproduction traits. AA genotypes yielded more milk (2332.34 kg, p = 0.04) with higher fat% (6.10, p = 0.004) and fat yield (155.75 kg, p = 0.06), reach peak milk production at 42.19 days and required 2.19 services for conception in comparison with GG genotypes. Birthweight of animals with AA genotype was lesser than with GG genotype (39.35 and 43.67 kg, p = 0.02, respectively). The days open is numerically better in AA genotype animals (99.35 days), but the difference between the three genotypes was non‐significant. The distinct significant associations reported in this study suggested that Leptin is reputable candidate genetic marker, which might be used to enhance animals’ genetic potential for milk production in conjunction with reproduction.
- Overexpression of C/EBPβ Affects The Cell Cycle Regulators and
Spermatogenesis Related Genes Expression And Function of Bovine Sertoli
- Authors: K Tang; Y Jin, F Chen, L Wang
Abstract: CCAAT/enhancer‐binding protein beta (C/EBPβ), an important transcriptional factor, plays a pivotal role in the regulation of female germ cell development. However, the role of C/EBPβ on the development of male germ cells has not been reported. In this study, we constructed the recombinant adenovirus plasmids of bovine C/EBPβ gene and harvested the subsequent adenoviruses, and then assessed the mRNA levels of spermatogenesis‐related genes (real‐time PCR) and secretion of inhibin B after 48 h of Ad‐C/EBPβ recombinant adenovirus infection in bovine sertoli cells (SCs). We found that overexpression of exogenous C/EBPβ gene upregulated the mRNA expressions of spermatogenesis‐related genes, including Pdgfa, Claudin, Caspase‐3, Occludin, kit1 and Cyclin E, and decreased the mRNA levels of Cyclin D1. Meanwhile, overexpression of exogenous C/EBPβ gene significantly increases the amounts of secreted inhibin B. In conclusion, the results indicate that the C/EBPβ gene plays an important regulatory role in regulation of the cell cycle regulators and spermatogenesis‐related genes expression and function of bovine SCs.
- Lysophosphatidic Acid Synthesis and its Receptors’ Expression in the
Bovine Oviduct During the Oestrous Cycle
- Abstract: Lysophosphatidic acid (LPA) is a naturally occurring simple phospholipid which in the bovine reproductive system can be produced in the endometrium, corpus luteum, ovarian follicle and embryo. In this study, we examined the possibility that LPA receptors are expressed, and LPA synthesized, in the bovine oviduct. We found that the concentration of LPA was highest in infundibulum in the follicular phase of the oestrous cycle and was relatively high during the early‐luteal phase in all examined parts of the oviduct. We also documented that LPA synthesis engages both available pathways for LPA production. The autotaxin (ATX) protein expression was significantly higher in the infundibulum compared to the isthmus during the follicular phase of the oestrous cycle. During the early‐luteal phase of the oestrous cycle, ATX and phospholipase A2 (PLA2) protein expression was highest in ampulla, although the expression of LPARs was not as dynamic as LPA concentration in the oviduct tissue, and we presume that in the bovine oviduct, the most abundantly expressed receptor is LPAR2. In conclusion, our results indicate that the bovine oviduct is a site of LPA synthesis and a target for LPA action in the bovine reproductive tract. We documented that LPAR2 is the most abundantly expressed in the bovine oviduct. We hypothesize that in the bovine oviduct, LPA may be involved in the transport of gametes, fertilization and cellular signalling between the oviduct and cumulus–oocyte complex.
- The Central Effect of β‐Endorphin and Naloxone on The
Biosynthesis of GnRH and GnRH Receptor (GnRHR) in The
Hypothalamic‐Pituitary Unit of Follicular‐Phase Ewes
- Abstract: The effects of prolonged, intermittent infusion of β‐endorphin or naloxone into the third cerebral ventricle of follicular‐phase ewes on the expression of genes encoding GnRH and GnRHR in the hypothalamus and GnRHR in the anterior pituitary gland (AP) were examined by an enzyme‐linked immunoabsorbent assay. Activation or blockade of μ‐opioid receptors significantly decreased or increased the GnRH concentration and GnRHR abundance in the hypothalamus, respectively, and affected in the same way GnRHR quantity in the AP gland. The changes in the levels of GnRH and GnRHR after treatment with β‐endorphin as well as following action of naloxone were reflected in fluctuations of plasma LH concentrations. On the basis of these results, it is suggested that β‐endorphinergic system in the hypothalamus of follicular‐phase ewes affects directly or via β‐endorphin‐sensitive interneurons GnRH and GnRHR biosynthesis leading to suppression in secretory activity of the hypothalamic‐pituitary axis.
- Changes in the Expression of the Prolactin Receptor (PRLR) Gene in
Different Physiological Stages in the Mammary Gland of the Iranian Adani
- Authors: S Morammazi; AA Masoudi, R Vaez Torshizi, A Pakdel
Abstract: The actions of prolactin hormone are mediated by prolactin receptor (PRLR), and proliferation and differentiation of secretory mammary epithelium are dependent on the presence of its receptors. To understand the PRLR expression pattern in mammary gland of dairy goat during different lactation stages, in this study, we first estimated the milk yield breeding value by multitrait random regression model and then compared the expression of the gene in different physiological stage of mammary gland between high‐ and low‐breeding value groups. We assayed the transcription level of the gene by quantitative real‐time PCR method, and its outcomes were analysed by a statistical model containing breeding value groups, sampling times and their interactions as fixed effects. The results indicated that the expression levels of PRLR gene were significantly upregulated in the drying stage (p
- Comparison of DNA Fragmentation Assay in Frozen‐Thawed Cat
- Abstract: DNA fragmentation of frozen‐thawed feline epididymal sperm from corpus and cauda regions was evaluated by three different techniques. The DNA fragmentation index (DFI) was compared between techniques: the sperm chromatin structural assay (SCSA®), acridine orange staining techniques (AOT) and the sperm chromatin dispersion (SCD). There were significant differences in DFI among the techniques (p
- Equine Chorionic Gonadotropin and Human Chorionic Gonadotropin Stimulation
Increase the Number of Luteinized Follicles and the Progesterone Level
Compared with Cabergoline Stimulation in Anoestrus Bitches
- Abstract: In this study, ovarian morphologies and blood progesterone concentrations following oestrous induction in bitches were examined. Fifty‐three clinically healthy anoestrus bitches received cabergoline at a daily dose of 5 μg/kg of body weight per os for 21 days (group I) or subcutaneous equine chorionic gonadotropin at a dose of 20 IU/kg of body weight for five consecutive days with an additional 500 IU s.c. per bitch of human chorionic gonadotropin on the last day of treatment (group II). Twenty bitches that spontaneously displayed oestrous signs were left untreated and served as controls (group III). The induced oestrous rates and ovulation rates in groups I and II were 60.0% vs 64.3% and 86.7% vs 83.3%, respectively. Morphological assessments of the ovarian structures after ovariohysterectomy revealed an increase in the number of luteinized follicles and cysts in group II compared with the two other groups (p
- Effect of Different Levels of Silymarin and Caproic Acid on Storage of Ram
Semen in Liquid Form
- Abstract: Two experiments were designed to evaluate the effect of silymarin on stored spermatozoa using four rams. In experiment 1, silymarin was evaluated as a supplement for Tris–glucose extender. Semen samples (n = 20) were diluted with extender containing 0, 50, 100, 150 and 200 μg/ml silymarin and incubated at 5°C for 72 h. Membrane integrity, acrosome integrity, sperm viability and motility were evaluated at 72 h. Concentration of malondialdehyde (MDA) was determined after 48 h. Membrane integrity was higher in 100 μg/ml silymarin (65.2%) than control group (43.2%, p
- Detection and isolation of Toxoplasma gondii from fresh semen of naturally
infected dogs in Southern Brazil
- Abstract: The aim of this study was to isolate Toxoplasma gondii and determine the viability of the parasite in fresh semen samples of clinically healthy adult dogs naturally infected. Eleven seropositive dogs with T. gondii IgG antibodies from southern Brazil were selected to confirm the presence and viability of T. gondii in fresh semen samples using in vitro isolation in Vero cell culture, polymerase chain reaction (PCR) and sequencing analysis. The presence of viable T. gondii was confirmed by in vitro isolation and PCR in five semen samples. The ITS1 region of the isolated protozoa (TG S4) was amplified and sequenced. The nucleotide sequence obtained was 99% compatible with the T. gondii DNA sequences stored in the GenBank. It has been shown that T. gondii tachyzoites may be isolated in vitro from fresh semen samples of clinically healthy dogs seropositive for T. gondii.
- Comparison of Intrauterine Antibiotics versus Ozone Medical Use in Sheep
with Retained Placenta and Following Obstetric Assistance
- Abstract: This study outlines a new approach to reproductive tract treatment using ozone foam spray for certain ovine obstetrical problems, such as retained foetal membranes and possible uterine infections following obstetric assistance (OA), in comparison with classical antibiotics treatments. The study was conducted on 256 ewes from 11 sheep farms in north‐western Croatia. A total of 139 ewes were diagnosed with dystocia (DT) and 49 with retention of placenta (RP). Ewes with RP were treated either with ozone foam spray (Riger spray G; Novagen®) applied into the body of the uterus for 2–3 s (first or RPO group; n = 24) or with two foaming, intrauterine tablets of oxytetracycline hydrochloride (Geomycin® F) (second or RPA group; n = 25). The third and fourth groups consisted of ewes that received OA for dystocia (including ringwomb, foetal oversize and assistance of abnormal position and posture). The third group (DTO; n = 70) was treated with ozone foam spray, while ewes in fourth group (DTA; n = 69) were treated with antibiotics. The ewes in the control group (CTL) with physiological puerperium were randomly selected (n = 70) from all herds. Transrectal ultrasonography (transversal diameter of uterine horns) was used for the control of uterus regression on days 2 and 25 after parturition. There was a difference in transversal uterine horn diameter in the RP groups, that is RPO and RPA (5.40 ± 0.53 cm vs. 5.43 ± 0.40 cm), ewes with dystocia, that is DTO and DTA (5.37 ± 0.49 cm vs. 5.54 ± 0.60 cm) and ewes from the CTL group (4.98 ± 0.35 cm) one day after parturition. Average transversal uterine diameter of all groups at day 25 post‐partum was 1.80 ± 0.15 cm. The intrauterine ozone treatment in ewes with RP and after manual obstetrics attained similar results to spontaneously delivered ewes (CTL group), showed as the physiological regression of the uterus with a similar transversal diameter without the presence of lochia in the uterine lumen, indicating that this could be a novel potential alternative therapy.
- Buffalo (Bubalus bubali) Late Embryo and Foetus Development: A
- Abstract: Many researches describe the embryonic developmental features in domestic animals; however, in farm animals, they are scarce. Most farm animal studies are related to assisted reproduction and embryos transfer techniques. But, morphological features and size measure to estimate the age gestation are rarely reported in literature. Thus, in this study, we described the developmental changes in the bubaline (Bubalus bubali) concepts from 21 to 60 days of gestation. Our results revealed that buffalo embryos similar morphological characteristics similar to other mammalian species. Also, similarities between bovine and bubaline persist; except on foetal stages when buffalos have a faster development than bovine. Therefore, buffalo's gestation period exhibits some varieties and accurate embryo age is more difficult. Yet, when we use a combination of the crown–rump, macroscopic analysis and alizarin red, it is possible to describe better the whole embryogenesis stages of the buffalo and which can contribute for future reproduction researches and applications in veterinary practice.
- Protective Effects of Quercetin on Selected Oxidative Biomarkers in Bovine
Spermatozoa Subjected to Ferrous Ascorbate
- Abstract: Quercetin (QUE) is a natural flavonol‐type flavonoid with antibacterial, anti‐inflammatory and anti‐aggregatory properties. It is also a powerful reactive oxygen species (ROS) scavenger and chelating agent. The aim of this study was to assess the effectiveness of QUE to reverse ROS‐mediated alterations to the motility, viability and intracellular antioxidant profile of bovine spermatozoa. Spermatozoa were washed out of fresh bovine semen, suspended in 2.9% sodium citrate and subjected to QUE treatment (7.5, 25, 50 and 100 μmol/l) in the presence or absence of a pro‐oxidant, that is ferrous ascorbate (FeAA; 150 μmol/l FeSO4 and 750 μmol/l ascorbic acid) during a 6‐h in vitro culture. Spermatozoa motion characteristics were assessed using the SpermVision computer‐aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified via luminometry, and the nitroblue tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the in vitro culture to investigate the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). FeAA treatment led to a reduced sperm motility (p
- Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine
- Authors: CG Luchetti; RJ Bevacqua, MS Lorenzo, MF Tello, M Willis, CP Buemo, DM Lombardo, DF Salamone
Abstract: The use of vesicles co‐incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co‐incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx‐egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p
- Seasonal and Ageing‐Depending Changes of Aquaporins 1 and 9
Expression in the Genital Tract of Buffalo Bulls (Bubalus bubalis)
- Authors: S. Arrighi; G. Bosi, G. Accogli, S. Desantis
Abstract: The presence of Aquaporins 1 (AQP1) and 9 (AQP9), integral membrane water channels that facilitate rapid passive movement of water and solutes, was immunohistochemically detected in the excurrent ducts collected from sexually mature buffalo bulls of proven fertility during the mating (late autumn–winter) and non‐mating (late spring to the beginning of autumn) seasons. Furthermore, the research was performed also on the epididymal cauda of a senile buffalo bull with inactive testis. Aquaporins 1 and 9 were immunolocalized at distinct levels. In the efferent ducts, AQP1 immunoreactivity was strongly evidenced at the apical surface of the non‐ciliated cells and weakly along the basal membrane of the epithelial cells. The latter reactivity disappeared during the non‐mating season. No AQP1 immunoreactivity was detected in the epithelium of epididymis and vas deferens, whereas AQP1 was expressed in the smooth muscle layer of the vas deferens. Aquaporin 1 was present in the blood vessels and in small nerve bundles all along the genital tract. The supranuclear zone of the epididymal principal cells was AQP9 immunoreactive, limited to the corpus and cauda regions, and vas deferens. The samples collected in the two reproductive seasons showed a weaker AQP9 immunoreactivity during the non‐mating season. A typical AQP9 immunoreactivity was noticed in the old buffalo examined. The tested AQP molecules showed a different expression pattern in comparison with laboratory mammals, primates, equine, dog and cat. In addition, seasonal differences were noticed which are possibly useful in regard to the comprehension of the morphophysiology of reproduction in the bubaline species, which are still a matter of debate.
- Sperm Morphology Assessment in Captive Neotropical Primates
- Abstract: The main objective of this study was to evaluate sperm morphology in four neotropical primate species to compare the sperm morphological traits and the sperm morphometric parameters as a basis for establishing normative sperm standards for each species. Data from 80 ejaculates collected from four primate species, Callithrix jacchus, Callimico goeldii, Alouatta caraya and Ateles geoffroyi, were analysed for detection of sperm morphological alterations using subjective World Health Organization (WHO‐2010) standards and Sperm Deformity Index (SDI) criteria, objective computer‐assisted sperm morphometry analysis (CASMA) and subpopulation sperm determination (SSD) methods. There were multiple differences (p
- Fusion of Boar Sperm with Nanoliposomes Prepared from Synthetic
- Authors: VR Kasimanickam; MM Buhr
Abstract: Liposomes are artificial membrane vesicles that can be used to test and model the functions and interactions of various biological membranes, or as a carrier system to deliver biologically active substances into the cells, or to incorporate lipids into the plasma membrane of target cells to modify membrane structure–function relationships. Sperm plasma membrane undergoes lipid modification during maturation in epididymis and during capacitation in the female reproductive tract to facilitate fertilization. Natural variation in the amounts and composition of lipids in the sperm plasma membrane may also contribute to the species‐specific sperm sensitivities to handling and storage conditions. Boar sperm are notoriously susceptible to membrane damage and are resistant to compositional alteration by artificial liposomes. This study used flow cytometry to demonstrate stable incorporation of nanoliposomes prepared from a complex mixture of various phospholipids (phosphatidylcholine : phosphatidylethanolamine : sphingomyelin : phosphatidylserine : phosphatidylinositol) with high fusion efficiency. Over 90% of sperm rapidly took up fluorescently labelled liposomes and retained the lipids for at least 60 min, in a significant time‐ and concentration‐dependent manner. This unique fusion efficacy could be used to alter sperm plasma membrane composition and hence membrane‐based functional responses.
- Pregnancy Loss in Dairy Cattle: Relationship of Ultrasound, Blood
- Abstract: Objectives were to determine associations between percentage pregnancy loss (PPL) in dairy cattle and: (i) pregnancy diagnosis by ultrasonography; (ii) pregnancy diagnosis by serum pregnancy‐specific protein B (PSPB) concentrations, with or without serum progesterone concentrations; and (iii) production and environmental factors. This study included 149 822 pregnancy diagnoses conducted over 13 years in Holstein‐Friesian cows in Hungarian dairy herds. The following were determined: PPL in cows diagnosed pregnant by transrectal ultrasonography 29–42 days after artificial insemination (AI; n = 11 457); PPL in cows diagnosed pregnant by serum PSPB 29–35 days after AI (n = 138 365); and PPL and its association with serum progesterone concentrations, PSPB and production/environmental variables. The definition of PPL was percentage of cows initially diagnosed pregnant based on ultrasonography or PSPB, but not pregnant when examined by transrectal palpation 60 –70 days after AI. The PPL was lower (p
- First Production of Larvae Using Cryopreserved Sperm: Effects of
Preservation Temperature and Cryopreservation on European Eel Sperm
- Abstract: Sperm cryopreservation is a useful tool in captive fish reproduction management, that is to synchronize gamete production, especially in the case of species as the European eel, where the time of female spawning readiness is unpredictable. Several protocols to cryopreserve sperm of this species have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization trials to produce viable offspring. In our experiment, the sperm motility was evaluated after the extraction and the best samples were selected and pooled. Until stripping of eggs and fertilization, diluted sperm samples were maintained at either 4 or 20°C, or cryopreserved, following existing protocols. Fertilization of two egg batches was attempted. Diluted sperm caused a similar percentage of fertilized eggs and a similar number of embryos and larvae, independently of storage temperature (4 or 20°C). The cryopreserved sperm resulted in a lower percentage of fertilized eggs, but embryos developed and a few larvae (‘cryolarvae’) were obtained 55 h after fertilization in one of the two egg batches. This result evidences that the tested cryopreservation protocol is applicable for eel reproduction management, although improvements will be required to enhance fertilization success.
- Serum MX2 Protein as Candidate Biomarker for Early Pregnancy Diagnosis in
- Authors: L Buragohain; R Kumar, T Nanda, SK Phulia, AK Mohanty, S Kumar, S Balhara, SPS Ghuman, I Singh, AK Balhara
Abstract: Interferon‐tau (IFN‐τ)‐induced molecular markers such as ubiquitin‐like modifier (ISG15), 2′,5′‐oligoadenylate synthetase 1 (OAS1) and myxovirus resistance genes (MX1 and MX2) have generated immense attention towards developing diagnostic tools for early diagnosis of pregnancy in bovine. These molecules are expressed at transcriptional level in peripheral nucleated cells. However, their presence in the serum is still a question mark. This study reports sequential changes in expression of MX2 transcript in whole blood and serum MX2 protein level on days 0, 7, 14, 21, 28 and 35 in pregnant (n = 9) buffalo heifers, and on days 0, 7 and 14 in non‐inseminated (n = 8) and inseminated non‐pregnant (n = 10) control animals. In non‐inseminated and inseminated non‐pregnant heifers, the differential expression of MX2 transcript and MX2 protein level remained similar between day 7 and 14 post‐oestrus. However, in pregnant heifers, on 14th and 28th day post‐insemination MX2 transcript was 16.38 ± 1.57 and 28.16 ± 1.91 times upregulated as compared to day 0. Similarly, serum MX2 protein concentration followed analogous trend as MX2 transcript and increased gradually with the progression of pregnancy. Correlation analysis between expression of MX2 transcript and its serum protein level showed a significant positive correlation in pregnant animals, while it was random in other two groups. Therefore, MX2 surge at transcriptional and serum protein level after day 14–28 of pregnancy in buffalo holds potential for its use in early pregnancy detection.
- Can Microfiltered Seminal Plasma Preserve the Morphofunctional
Characteristics of Porcine Spermatozoa in the Absence of Antibiotics?
A Preliminary Study
- Authors: F Barone; D Ventrella, A Zannoni, M Forni, ML Bacci
Abstract: Artificial insemination is extensively performed in pig farms in Europe, the United States and Canada. Antibiotics are typically added to the inseminating dose to limit bacterial growth during liquid phase storage at 16°C, as bacterial contamination is unavoidable. The World Organization for Animal Health (OIE) and the Food and Agriculture Organization (FAO) take action to control and reduce antibiotic use in animals as more bacteria are becoming resistant to antimicrobials. To avoid the use of antibiotics, we prepared inseminating doses using microfiltered seminal plasma (SP). Microfiltration is a common technology used to reduce bacterial contamination but may retain seminal substances, influencing sperm quality during storage. Therefore, the aim of this study was to evaluate the morphofunctional parameters of spermatozoa during storage at 16°C in doses prepared with or without microfiltered SP, with or without the addition of antibiotics, in a Latin square design. Artificial insemination doses with microfiltered SP and without antibiotic addition preserved spermatozoa viability, mitochondrial membrane potential, acrosome integrity and objective motility, with absolute values equal or even better than those observed in conventional doses. In conclusion, although the results could be considered preliminary due to the small sample size, this study suggests that microfiltration of SP can be a simple method, feasible on farms, to replace antibiotic use in extended doses stored in the liquid phase at 16°C for up to 7 days.
- Effects of Inulin Supplementation in Low‐ or High‐Fat Diets on
Reproductive Performance of Sows and Antioxidant Defence Capacity in Sows
- Authors: YS Wang; P Zhou, H Liu, S Li, Y Zhao, K Deng, DD Cao, LQ Che, ZF Fang, SY Xu, Y Lin, B Feng, J Li, D Wu
Abstract: This experiment was conducted to investigate the effects of inulin supplementation in low‐ or high‐fat diets on both the reproductive performance of sow and the antioxidant defence capacity in sows and offspring. Sixty Landrace × Yorkshire sows were randomly allocated to four treatments with low‐fat diet (L), low‐fat diet containing 1.5% inulin (LI), high‐fat diet (H) and high‐fat diet containing 1.5% inulin (HI). Inulin‐rich diets lowered the within‐litter birth weight coefficient of variation (CV, p = 0.05) of piglets, increased the proportion of piglets weighing 1.0–1.5 kg at farrowing (p
- Effect of Ca Ionophore On Blastocyst Production Following Intracytoplasmic
Sperm Injection in Caprine Oocytes
- Authors: SD Kharche; J Pathak, S Agarwal, B Kushwah, AKS Sikarwar
Abstract: The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro‐matured Caprine oocytes. A total of 470 in vitro‐matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48–72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro‐matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm‐injected in vitro‐matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non‐activated oocytes.
- Impact of Buserelin Acetate or hCG Administration on the Day of First
Artificial Insemination on Subsequent Luteal Profile and Conception Rate
in Murrah Buffalo (Bubalus bubalis)
- Authors: AK Pandey; SPS Ghuman, GS Dhaliwal, SK Agarwal, JB Phogat
Abstract: This study was designed to investigate the impact of buserelin acetate (BA) or human chorionic gonadotropin (hCG) administration on the day of first artificial insemination (AI) on subsequent luteal profile (diameter of corpus luteum (CL) and plasma progesterone) and conception rate in Murrah buffalo. The present experiment was carried out at two locations in 117 buffalo that were oestrus‐synchronized using cloprostenol (500 μg) administered (i.m.) 11 days apart followed by AI during standing oestrus. Based on treatment (i.m.) at the time of AI, buffalo were randomly categorized (n = 39 in each group) into control (isotonic saline solution, 5 ml), dAI‐BA (buserelin acetate, 20 μg) and dAI‐hCG (hCG, 3000 IU) group. Out of these, 14 buffalo of each group were subjected to ovarian ultrasonography on the day of oestrus to monitor the preovulatory follicle and on days 5, 12, 16 and 21 post‐ovulation to monitor CL diameter. On the day of each sonography, jugular vein blood samples were collected for the estimation of progesterone concentrations. All the buffalo (n = 117) were confirmed for pregnancy on day 40 post‐ovulation. The conception rate was better (p
- Influence of Glutamine Supplementation on Motility and Fertilization
Success of Frozen–Thawed Persian Sturgeon (Acipenser persicus) Sperm
- Authors: MS Aramli; K Golshahi, RM Nazari, A Golpour, S Aramli
Abstract: Amino acids have an important biological role for the prevention of cell damage during cryopreservation. The aim of this study was to investigate the effects of glutamine on post‐thaw sperm motility and fertilization success in the Persian sturgeon (Acipenser persicus). Sperm collected from six fish was cryopreserved in extenders containing different glutamine concentrations (2.5, 5 and 10 mm). Sperm samples diluted at the ratio of 1 : 1 using the extenders were subjected to cryopreservation. After dilution, the sperm suspensions were sucked into 250‐μl straws; the straws were placed on the tray, frozen in nitrogen vapour and plunged into liquid nitrogen. Then, sperm were thawed in a water bath at 40°C for 5 s and used for analysis. Our results revealed that an increase in the concentration of glutamine caused a significant increase in the motility percentage, curvilinear velocity (VCL) and also fertilization success in the Persian sturgeon (p
- Issue Information
- Pages: 451 - 452