Testicular dysfunction is often associated with an imbalance in antioxidant/oxidant homeostasis, leading to negative effects of oxidative stress on germ cell proliferation, steroidogenesis, and sperm function. Trace minerals (TM) are involved in basic homeostatic and enzymatic processes like free radical detoxification, cellular respiration, carbohydrates, lipids, nucleic acids, synthesis and metabolism of proteins, and stabilization of membranes and DNA. Dietary source of TM is necessary to support these processes. Supplementing cattle with TM is nowadays a common practice to help support growth, reproduction, and immunity. This review provides information on roles of TM in bulls testicular and sperm functions, and effects of TM mineral supplementation on bull reproductive parameters.

The objectives of this study were to validate diagnostic tests to detect polymorphonuclear cells (PMNs) in bull semen, and to determine the prevalence of leucospermia in beef bulls with varying semen quality. We hypothesized that all tests have comparable diagnostic value, and that leucospermia is more prevalent in unsatisfactory breeders in association with poor semen quality. For the analytical validation, one ejaculate was obtained from five bulls. Aliquots of 50 x 10(6) purified sperm were incubated in triplicate with six concentrations of purified bovine PMNs: 1) no PMNs, 2) 0.25 x 10(6) PMN/ml, 3) 0.5 x 10(6) PMN/ml, 4) 2.5 x 10(6) PMN/ml, 5) 5 x 10(6) PMN/ml, 6) 10 x 10(6) PMN/ml. The PMNs were quantified using a hemacytometer, cytology, a leucocyte esterase dipstick test (LEDT), a peroxidase test, and CD45 immunolabeling. The number of leucocytes detected with the LEDT differed among treatments (P < 0.0001). The quantitative tests detected differences with the control treatment at a PMN concentration of >= 2.5 x 10(6) PMN/ml (P < 0.0001). Sperm motion parameters after 4 h of incubation at 38 degrees C were lower in samples with >= 5 x 10(6) PMN/ml (P < 0.05). For the clinical validation, semen samples from 305 beef bulls were evaluated. Unsatisfactory breeders (n = 83) had more CD45-positive cells (P = 0.016) and positive LEDT results (P = 0.008) than satisfactory breeders (n = 222). With CD45 immunostaining as the gold standard, the hemacytometer count had the highest clinical sensitivity (64.3 %) but the lowest specificity (73.3 %). A higher specificity was obtained with the peroxidase test (95.1 %) or semen cytology (98.8 %). In conclusion, the presence of >= 5 x 10(6) PMN/ml was associated with decreased semen quality in beef bulls. The hemacytometer count was the most sensitive bull-side test. But due to the low specificity, positive hemacytometer counts should be confirmed with the identification of peroxidase-positive cells or morphological identification of leucocytes on semen cytology. The CD45 immunostaining is the gold standard for the diagnosis of leucospermia in bulls.

Trace minerals (TM) have a crucial role in cattle reproduction. Although microelements are required in small amounts, their bioavailability is essential for the cow reproductive physiology, and for adequate fertility and productivity. They are particularly important for antioxidant protection against cellular damage (e.g. gametes and embryonic cells), hormone synthesis, and pregnancy maintenance. Oral TM supplementation is a common and highly recommended management practice in cattle operations. However, there is substantial variability in TM bioavailability in animals receiving oral TM supplementation. The strategic use of injectable TM supplementation (without replacing traditional oral TM supplements) before episodes of marked stress (e.g. parturition), higher metabolic demand with TM depletion (e.g. last trimester of pregnancy), active immune response (e.g. uterine involution or vaccination), and before breeding helps to maintain adequate TM and oxidative status during critical points of the reproductive program. This manuscript reviews the research-based evidence regarding the effects of TM supplementation on bovine reproduction and its impact on beef and dairy cattle reproductive performance.

No studies have evaluated the peripartum follicular dynamics resulting in foal heat under tropical environments. We aimed to assess retrospectively the peripartum follicular dynamics in Colombian Paso Fino mares that were inseminated at the foal heat, becoming pregnant or not. Records including follicular dynamics of pregnant mares prepartum and from foaling until foal heat ovulation were assessed in Colombian Paso Fino mares (CPF, n = 24) bred under permanent grazing in a tropical herd in Colombia. The number of ovarian follicles >10 mm before foaling and the largest follicle (F1) growth rate (mm/day) from foaling until the F1 reached the largest diameter (pre-ovulatory size) at the foal heat were assessed. Mares were inseminated at foal heat with 20 mL of semen (at least 500 million live spermatozoa) with >75% motility and 80% viability from a stallion of proven fertility. Ovulation was confirmed the day after follicles had reached the largest diameter. Quantitative data from follicular growth, the day at ovulation, from mares that became pregnant (PM) or not (NPM) at 16 days post-insemination were compared by one-way ANOVA, repeated measures ANOVA (follicle growth rate data) or Chi-square test (edema and cytology scores data). Epidemiological data, gestation length, and the number of follicles on third prepartum days did not significantly differ between PM and NPM (p > 0.05). Seventy-one percent of mares (17/24) got pregnant. Ovulatory follicles grew faster in the NPM group (n = 7), which ovulated between the seventh and ninth postpartum days, compared to PM (n = 17), which ovulated between the 11th and 13th postpartum days. Pre-ovulatory follicle diameter in PM (48.57 ± 0.8 mm) was significantly larger than in NPM (42.99 ± 1.0 mm) (p < 0.05). In addition, the PM edema score (2.93 ± 0.32 mm) on ovulation day was significantly lower (p < 0.05) than NPM (4.47 ± 0.05 mm). First postpartum ovulation occurred at 12.6 ± 0.3 and 8.5 ± 0.4 days (p < 0.05) in PM and NPM, respectively. Colombian Paso Fino mares bred under permanent grazing under tropical rainforest conditions with no foaling or postpartum complications showed a 71% gestation rate when inseminated at foal heat when ovulation occurs between the second and third postpartum week.

The objectives of this study were to evaluate the ability of five diagnostic tests to detect polymorphonuclear cells (PMNs) in stallion semen, and to determine the concentration of PMNs that affects sperm motility. We hypothesized that all tests have diagnostic value, and even low concentrations of PMNs affect motility. One ejaculate was obtained from six stallions. Aliquots of 50 x 10 6 purified sperm were incubated, in triplicate, with six concentrations of purified PMNs: 1) no PMNs, 2) 0.25 x 10 6 PMN/ml, 3) 0.5 x 10 6 PMN/ml, 4) 2.5 x 10 6 PMN/ml, 5) 5 x 10 6 PMN/ml, 6) 10 x 10 6 PMN/ml. The PMNs were quantified using a hemacytometer, cytology, a leucocyte esterase dipstick test (LEDT), a peroxidase test, and CD13 immunolabeling. Sperm motility was evaluated after 4 h at 38 degrees C. The number of leucocytes detected with the LEDT differed among treatments (P < 0.0 0 01), from negative results in control samples to moderate or large numbers in the samples with the highest PMN concentration. The hemacytometer count and CD13 immunostaining detected differences with the control treatment at the lowest PMN concentration (2.5 x 10 6 PMN/ml; P < 0.001). Sperm motion was lower in samples with >= 5 x 10 6 PMN/ml (P < 0.0 0 01). Thus, a sample was considered leucospermic if it contained >= 5 x 10 6 PMN/ml. The LEDT had the best sensitivity (10 0%), followed by cytology (78%), peroxidase test (60%), CD13 immunostaining (56%) and hemacytometer count (47%). The LEDT had the lowest specificity (65%), which was 95% for all other tests. In conclusion, the LEDT was a simple, economic and sensitive stall-side test to screen semen for presence of PMNs. Because of the lower specificity, positive LEDT results should be confirmed with the identification of peroxidase-positive cells or CD13-positive cells. (c) 2023 Elsevier Inc. All rights reserved.

Sperm-oocyte fusion is an essential function during the fertilization process. Membrane fusion occurs as a result of a complex series of intermolecular interactions between the sperm and the oocyte. Proteins involved in this interaction include izumo sperm-egg fusion 1 (IZUMO1), sperm acrosome associated 6 (SPACA6), transmembrane protein 95 (TMEM95), fertilization influencing membrane protein (FIMP) and sperm-oocyte fusion required 1 (SOF1), ADAM metallopeptidase domain 2 (ADAM2), cysteine rich secretory protein 1-3 (CRISP1-3), cation channel sperm associated 1-4 (CATSPER1-4) and polycystin family receptor for egg jelly (PKDREJ) (Rivera and Swanson, 2022. Front. Cell Dev. Biol., 10:827-827454) on the male gamete, and izumo 1 receptor (JUNO) and cluster of differentiation 9 (CD9) on the female gamete (Inoue et al., 2021. eLife; 10:e66313). The IZUMO1-JUNO interaction is essential for gamete fusion and the block of polyspermy (Bianchi et al., 2014: Nature, 508:483-487). Other proteins called dendrocyte expressed seven transmembrane protein (DC-STAMP) and homologue domain-containing 1 and 2 (DCST1/2) are also required for sperm-egg fusion in mice (Inoue et al., 2021. eLife; 10:e66313). The objective of this study was to identify and quantify the expression of genes associated with sperm-oocyte interaction during the fertilization process in tissue samples from the testis, head, body and tail of the epididymis. Tissues were collected after castration from three light-breed reproductively normal stallions(5-13 years). mRNA was extracted and libraries were generated using Next Generation Sequencing. FPKM of the genes of interest was compared between epididymal head, body and tail, and testis (control tissue) using in silico analysis (online databases). Expression of CRISP3, SPACA6, ADAM2, CATSPER1, CATSPER2, CATSPER3, CATSPER4, GLPR1L1, IZUMO1, PKDREJ, TMEM95, DCST1 and DCST2 genes were identified in the testis and all segments of the epididymis of the horse. All these genes, except for CRISP1, were up-regulated in the testis compared with the three segments of the epididymis, while CRISP1 was down-regulated in the testis compared with the epididymis. In this study, JUNO gene was expressed in the testis (FPKM 48.15), and was downregulated in the head, body and tail of the epididymis (FPKM 2.3, 3.2 and 2.9, respectively). In summary, genes encoding for proteins associated with sperm-oocyte interaction were more strongly expressed within the testis, suggesting that these proteins are acquired during spermatogenesis or spermiogenesis. Meanwhile, CRISP1 may be acquired during sperm transport along the epididymis. The expression of JUNO gene within the stallion reproductive tract is striking since the expression has not been reported in mice (RPKM 0) and human testis (RPKM 0.003). JUNO is a receptor only found on the oocyte membrane. The specific cells that express these genes, and their function within the stallion reproductive tract needs further studies.

Spermatogenesis and sperm maturation take place in the testes and epididymis, respectively. These complex physiological processes are regulated by multiple genes, which have not been studied in the stallion. In this study, Illumina sequencing was used to analyze the transcriptomes of the testes, head, body and tail of the epididymis from three reproductively normal stallions. The mRNA was extracted and the libraries were generated using Next Generation Sequencing (NGS). The expression patterns of differentially expressed genes (DEGs) in the testes and epididymis were determined by bioinformatics analysis. Gene Ontology (GO) enrichment analysis of DEGs was implemented and gene length bias was corrected. Differentially expressed genes were compared between the different segments of the epididymis and the testis (control tissue). GO-terms with adjusted p < 0.05 were considered significantly enriched. Among the four libraries, 18,732 genes were identified. Of those, 6,574 genes (39.1%) were tissue-specific. The distribution of tissue-specific genes was 14.2 % in the testes, 2.2 % in the epididymal head, 1.1 % in the epididymal body, and 1.4 % in the epididymal tail. Compared with testicular tissue, there were 13,349 DEGs (5,595 up-regulated and 7,754 down-regulated) in the epidydimal head, 14,207 DEGs (5,884 up-regulated and 8,323 down-regulated) in the epididymal body, and 14,822 DEGs (5,842 up-regulated and 8,980 down regulated) in the epididymal tail. In the epididymal head,the main downregulated terms were male gamete generation (224 DEGs), spermatogenesis (224 DEGs), organelle fission (264 DEGs), sexual reproduction (343 DEGs) and nuclear division (233 DEGs). The main upregulated terms were associated with the ribosome (322 DEGs), lymphocyte activation (226 DEGs), and cytosolic part (104 DEGs). In the epididymal body, the most significantly downregulated terms were male gamete generation (232 DEGs), spermatogenesis (232 DEGs), sexual reproduction (358 DEGs), cilium morphogenesis (151 DEGs) and cilium (235 DEGs). The most significantly upregulated terms were lysosome (150 DEGs), lytic vacuole (150 DEGs), positive regulation of locomotion (197 DEGs), cytosolic ribosome (71 DEGs), vacuole (182 DEGs). In the epididymal tail, the most significantly downregulated terms were male gamete generation (239 DEGs), spermatogenesis (193 DEGs), cilium (253 DEGs), ciliary part (186 DEGs), cilium morphogenesis (158 DEGs). The most significantly upregulated terms were associated with the ribosome (438 DEGs) and cytosolic part (109 DEGs). In summary, we found decreased expression of genes involved in gamete production, and increased expression of genes involved in protein synthesis, immunity and sperm transport in different segments of the epididymis. These findings provide genome-wide mRNA expression profiles for the horse epididymis and testes, providing a biological basis for future research on stallion fertility.

In brief Paternal high-gain diet reduces blastocyst development following in vitro fertilization and embryo culture but does not affect gene expression or cellular allocation of resultant blastocysts. Abstract Bulls used in cattle production are often overfed to induce rapid growth, early puberty, and increase sale price. While the negative consequences of undernutrition on bull sperm quality are known, it is unclear how a high-gain diet influences embryo development. We hypothesized that semen collected from bulls fed a high-gain diet would have a reduced capacity to produce blastocysts following in vitro fertilization. Eight mature bulls were stratified by body weight and fed the same diet for 67 days at either a maintenance level (0.5% body weight per day; n = 4) or a high-gain rate (1.25% body weight per day; n = 4). Semen was collected by electroejaculation at the end of the feeding regimen and subjected to sperm analysis, frozen, and used for in vitro fertilization. The high-gain diet increased body weight, average daily gain, and subcutaneous fat thickness compared to the maintenance diet. Sperm of high-gain bulls tended to have increased early necrosis and had increased post-thaw acrosome damage compared with maintenance bulls, but diet did not affect sperm motility or morphology. Semen of high-gain bulls reduced the percentage of cleaved oocytes that developed to blastocyst stage embryos. Paternal diet had no effect on the number of total or CDX2-positive cells of blastocysts, or blastocysts gene expression for markers associated with developmental capacity. Feeding bulls a high-gain diet did not affect sperm morphology or motility, but increased adiposity and reduced the ability of sperm to generate blastocyst-stage embryos.

Reproductive control of wild horses has frustrated land management groups for decades. GnRH and Zona Pellucida vaccines have a limited duration of efficacy, intrauterine devices are unpredictably retained, and attempts to ovariectomize mares in the western United States were met with public outcry in 2018. The development of a non-surgical permanent sterilization technique has the potential to revolutionize management of wild equids on public lands. The objectives of this study were to develop a safe and effective sterilization technique for reproductively healthy mares. We hypothesized that laser ablation of the oviductal papillae would be an effective method of permanent sterilization. Seven light-breed reproductively healthy mares (5-21yrs) were enrolled in the study. On enrollment, mares were sedated for hysteroscopy, and the endometrium and oviductal papillae were examined using a 103cm flexible endoscope (Olympus GIF-160 Gastroscope, Center Valley, PA) attached to an Olympus EVIS EXERA CV-160 video processor. In five mares, a 600 μm laser fiber was advanced through the biopsy channel and a diode laser (Dornier Medilas D, Dornier MedTech America, Inc., Kenneasaw, GA) was used to deliver direct contact pulses of 3 to 5 seconds in duration to the oviductal papilla. The endoscope was then guided up the contralateral horn and the process repeated. In two control mares, the oviductalpapillae were visualized, but no laser ablation was performed. All mares received 5 mg dinoprost IM and a uterine lavage within 4 hours post-procedure. Transrectal ultrasound was performed every 2-3 days until a 35mm follicle was detected, and then mares were bred using semen from a fertile stallion. Pregnancy status was determined by transrectal ultrasound 14 days post-ovulation, luteolysis was induced, and mares were rebred on 2-4 consecutive cycles. Control mares conceived on 6 out of 9 cycles (67% pregnancy rate). Pregnancy rate was significantly lower in the treatment group (5%, 1 out of 20 cycles, p=0.003). The first mare that was laser ablated conceived on the 4th cycle, and repeat hysteroscopy determined that the left oviduct was not effectively ablated. The third mare was diagnosed with peritonitis via abdominocentesis 6 hours post hysteroscopy. The mare was treated with broad spectrum antibiotics and recovered uneventfully. Three mares were re-bred two years after the procedure and did not conceive. In conclusion, when laser ablation of the oviductal papillae was appropriately performed, scar tissue formation effectively prevented pregnancy for a minimum of four months post-procedure. Laser ablation of the oviductal papillae is a promising technique for permanent sterilization of the mare and a follow-up long-term fertility study is warranted in a larger group of mares is warranted.

Uterine involution, ovarian activity, and incidence of postpartum uterine disease (PUD) were assessed in forty-eight dairy cows from calving until the 10th postpartum week. Postpartum follow-up included evaluation of uterine involution and ovarian structures by B-mode, Doppler color, and Doppler spectral ultrasound of the right uterine artery in cows with no calving or postpartum uterine problems (healthy cows). Data from cows that developed PUD (PUD cows) were compared with healthy cows matched by herd and days in milk (DIM). Data were analyzed by descriptive statistics, simple regression, one-way ANOVA, or repeated ANOVA measures, and in data analysis of healthy cows, uterine horn diameter assessed by B-mode ultrasound ranged from 22.9 ± 2.4 to 19.4 ± 1.4 mm and 19.9 ± 2.2 to 20.5 ± 2.3 mm from the fourth to the seventh postpartum week in the left and right uterine horns, respectively (P > 0.05). During the study, 15 and 7 cows had corpus luteum in the left and right ovaries, respectively. The mean time for the first postpartum CL was 30.1 ± 3.2 DIM (min 8, max 67 DIM). In data analysis of PUD cows, uterine blood flow assessed by color Doppler ranged from 7.4 ± 4.0 to 43.75 ± 10.3% in cows that developed PUD compared to 16.7 ± 11.0% in healthy cows (P > 0.05). No statistically significant changes were found in resistance index, pulsatility index, time-averaged maximum velocity, time-averaged mean velocity, or diastole/systole ratio (D/S) in cows that developed PUD compared to healthy cows (P > 0.05). Finally, no correlation was found between Doppler spectral parameters and uterine involution (P > 0.05). Our data suggest that cows receiving transition diets and exhibiting normal calving undergo a rapid macroscopic uterine involution and ovarian follicular dynamics resumption. Complete ultrasound evaluation provides valuable data for assessing uterine involution in postpartum dairy cows.

The lack of standardized tests and interpretative criteria makes the diagnosis of stallion leucospermia difficult. Our objectives were to evaluate the ability of six diagnostic tests to detect polymorphonuclear cells (PMNs) in stallion semen, and to determine the concentration of PMNs that affects sperm motility. We hypothesized that all tests have diagnostic value, and that low concentrations of PMNs affect sperm motility. One ejaculate was obtained from six stallions. Aliquots of 50 ×106 purified sperm from each stallion were incubated with six concentrations of purified PMNs: 1) no PMNs, 2) 0.25 ×106 PMN/ml, 3) 0.5 ×106 PMN/ml, 4) 2.5 ×106 PMN/ml, 5) 5 ×106 PMN/ml, and 6) 10 ×106 PMN/ml. Each treatment was performed in triplicate. The PMNs were quantified using a hemacytometer (phase contrast microscopy), cytology (light microscopy), a commercial leucocyte esterase test (dipstick), a commercial peroxidase test (light microscopy), and immunolabeling of the neutrophil marker CD13 (flow cytometry). Sperm motility was evaluated after 4 h at 38 °C. The frequency of results obtained with the esterase test differed among treatments (P<0.0001). Negative or trace results were observed only with ≤ 0.5 ×106 PMNs/ml. A small number was observed in intermediate samples. In samples with ≥ 5 ×106 PMN/ml, only moderate or large results were obtained. Among quantitative tests, the hemacytometer count and flow cytometry detected differences with the control treatment at the lowest PMN concentration (2.5 ×106 PMN/ml; P<0.001). Total and progressive sperm motility were lower in samples with ≥5 ×106 PMN/ml than in those with lower concentrations (P<0.0001). Thus, a sample was considered leucospermic with ≥ 5 ×106 PMN/ml. The leucocyte esterase dipstick test had the best sensitivity (100%), followed by cytology (78%), peroxidase test (60%), flow cytometry (56%) and hemacytometer count (47%). The esterase test had the lowest specificity (65%), whereas the specificity of all other tests was 95%. In conclusion, presence of ≥5 ×106 PMNs/ml was associated with impaired sperm motility. The leucocyte esterase dipstick test is a simple, economic and sensitive stall-side test to screen semen for presence of PMNs in quantities capable of affecting sperm motility. Because of the lower specificity, we recommend confirming positive esterase results with the identification of peroxidase-positive cells under light microscopy, or CD13-positive cells using flow cytometry.

The cytobrush is considered the method of choice to obtain endometrial samples. Rigid brush fibers, however, may induce endometrial irritation and bleeding, or cell fragmentation, decreasing quality and diagnostic value of the samples. It was hypothesized that samples collected using a novel cytotape would provide sample smears of greater quality and less blood contamination than the cytobrush. Endometrial samples were collected with a cytotape and a cytobrush from ten mares without endometritis. Endometritis was then induced with artificial insemination, and samples were again collected 6 h after insemination. A cytology smear and bacterial culture were prepared from each sample. The collection methods and times were compared in terms of number and integrity of endometrial cells; number, integrity, and percentage of neutrophils; number of red blood cells, and number of colony-forming units. Frequency of positive cytology and culture was compared when there was use of each technique. The sensitivity, specificity, positive predictive value, and negative predictive value of cytology and culture for each technique was calculated using endometrial biopsy as the gold standard. While all samples had adequate and comparable cellularity and cell integrity, cytotape samples had less red blood cell contamination compared to cytobrush samples (P < 0.05). The number and percentage of PMNs, frequency of positive cytology diagnosis, number of colony-forming units and frequency of positive cultures did not differ between collection methods. In conclusion, the cytotape is a rapid, easy, and practical technique that can provide endometrial samples with similar diagnostic value to the cytobrush, but with less blood contamination.

This study was performed to elucidate whether the route of booster vaccination affects the immune response against respiratory vaccine viruses in pre-weaning beef calves that receive primary intranasal (IN) vaccination during the first month of life. The objective was to compare the serum neutralizing antibody (SNA) titers to BHV1, BRSV, and BPI3V, cytokine mRNA expression and mucosal BHV1- and BRSV-specific IgA in nasal secretions following administration of IN or subcutaneous (SC) modified-live virus (MLV) booster vaccines 60 days after primary IN vaccination in young beef calves. Twenty-one beef calves were administered 2 mL of an IN MLV vaccine containing BHV1, BRSV, and BPI3V (Inforce3®) between one and five weeks of age. Sixty days after primary vaccination, calves were randomly assigned to one of two groups: IN-MLV (n = 11): Calves received 2 mL of the same IN MLV vaccine used for primary vaccination and 2 mL of a SC MLV vaccine containing BVDV1 & 2 (Bovi- Shield GOLD® BVD). SC-MLV (n = 10): Calves were administered 2 mL of a MLV vaccine containing, BHV1, BRSV, BPI3V, and BVDV1 & 2 (Bovi-Shield GOLD® 5). Blood and nasal secretion samples were collected on days -61 (primary vaccination), -28, -14, 0 (booster vaccination), 14, 21, 28, 42 and 60 for determination of SNA titers, cytokine gene expression analysis and nasal virus-specific IgA concentrations. Statistical analysis was performed using a repeated measures analysis through PROC GLIMMIX of SAS®. Booster vaccination by neither IN nor SC routes induced a significant increase in SNA titers against BHV1, BRSV, and BPI3V. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA titers (on day 42) and IgA concentration in nasal secretions (on days 21 and 42) compared to calves receiving IN booster vaccination. Both IN and SC booster vaccination were able to stimulate the production of BHV1-specific IgA in nasal secretions. In summary, booster vaccination of young beef calves using either SC or IN route two months after IN MLV primary vaccination resulted in comparable SNA titers, cytokine gene expression profile and virus-specific IgA concentration in nasal secretions. Only a few differences in the systemic and mucosal immune response against BHV1 and BRSV were observed. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA and secretory IgA titers compared to IN booster vaccination.

Different stallions exhibit a high level of variation in the ability of their sperm to survive cryopreservation. A large fraction of stallions show poor post-thaw sperm motility, and their semen is not suitable for commercial freezing. In this study, we hypothesized that the presence of sperm-bound antisperm antibodies (ASAs) was associated with poor cryosurvival of stallion sperm. Our objective was to assess the level of ASA binding to stallion sperm, and determine if it was associated with good or poor sperm cryosurvival. In Experiment 1, cooled shipped semen from 27 stallions was frozen using three commercial semen extenders. Sperm motility, membrane integrity, acrosome integrity and apoptosis were evaluated before and after freezing for each aliquot. In addition, the percentage of ASA-bound sperm was evaluated post-thaw. In Experiment 2, semen from 22 stallions was frozen immediately after collection a single formulation of semen extender. Sperm motility and ASA binding were evaluated post-thaw. The results of both experiments showed similar findings. The frequency of ASA-positive samples was higher among stallions with poor sperm cryosurvival (Exp. 1 and 2 = 6/11, 54.5%) than for good sperm cryosurvival (Exp. 1 = 0/16, 0%; Exp. 2 = 1/11, 9.1%). The percentage of IgG- and IgA-bound sperm was also higher in stallions with poor sperm cryosurvival in both experiments (P < 0.05). Post-thaw sperm motility, velocity and distance parameters were lower in ASA-positive than ASA-negative stallions (P < 0.005). No effect of the semen extender used was observed. In addition, stallions with ASAs had a higher percentage of apoptotic sperm than stallions without ASAs. The presence of sperm-bound ASAs was associated with poor cryosurvival for stallion spermatozoa. Thus, it may be beneficial to evaluate stallions for binding of ASAs prior to freezing to offer and indicator of the prognosis for cryosurvival.

While changes in semen quality after heat stress are well characterized in the bull, changes in endocrine function have not been critically evaluated. It was hypothesized here that scrotal insulation results in alterations in Sertoli cell and Leydig cell function, as measured by changes in serum testosterone and anti-Müllerian hormone (AMH) concentration. Scrotal insulation bags were placed in 10 bulls for 8 d. Blood was collected on days -22 and -2, and weekly from days 5 to 96 (day 0 = first day of scrotal insulation) for measurement of serum concentration of AMH and testosterone using ELISA. The concentration of AMH decreased on day 5, followed by an increase on day 54 (P = 0.014). When AMH concentration was normalized to pre-insulation values, the percent increase in serum concentration of AMH was significant between days 26 and 54, with another peak at 75 d (P = 0.031). The serum concentration of testosterone (P = 0.0001) and the percentage of change in testosterone concentration (P < 0.0001) increased on day 5, followed by a decrease from days 33 to 96. Scrotal insulation was associated with Sertoli and Leydig cell dysfunction, as measured by serum testosterone and AMH concentration. The persistently low concentration of testosterone at the end of the study suggests a long term effect of scrotal insulation on Leydig cell function.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

This study aimed to describe and compare semen parameters (pre-freeze and post-freezing) and anti sperm antibodies (ASA) of donkeys with epididymal sperm granuloma and healthy controls. Feral donkeys (n = 10) castrated in a concurrent study were enrolled in the present experiment. Three donkeys had unilateral granulomas, two donkeys had bilateral granulomas, whereas the remaining five had grossly normal epididymides. The granulomas were either single or multiple, firm, well-circumscribed, tan to red, and 1-5 mm in size. Upon incision, abundant, thick, tan to white-yellow fluid was recovered. Histopathology revealed epithelioid macrophages, multinucleated giant cells, and abundant sperm cell fragments with mineralized cellular debris. Each epididymis was dissected, and semen harvested for cryopreservation. Semen was assessed for sperm motility parameters, plasma membrane integrity, and mitochondrial membrane potential. All donkeys had semen cryopreserved in a standard manner. In addition, post-thaw semen from all donkeys was assessed for ASA (IgG and IgA), acrosome integrity and morphology. Post thaw, the progressive sperm motility and the percentage of sperm with an intact plasma membrane were reduced in donkeys with sperm granuloma ( P = 0.04). There was no difference in total sperm motility, morphology, or damaged acrosome across groups ( P > 0.05). Three donkeys with sperm granuloma (60%) displayed increased IgG and IgA ASA. In conclusion, sperm granulomas only marginally affected sperm quality and resulted in IgG ASA binding to sperm with damaged plasma membrane. It remains to be determined if sperm granuloma and ASA affect fertility in donkeys. (c) 2021 Elsevier Inc. All rights reserved.