Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

Metabolomic differences of seminal plasma between boars with high and low average conception rates after artificial insemination

Seminal plasma is a complex biological fluid containing many metabolites including amino acids, fructose, carbohydrates and lipids Metabolites play important roles in multiple biological processes, but details and significance of the seminal plasma metabolome related to boar fertility are unknown. The aim of the present study was to compare the comprehensive metabolome of seminal plasma from boars with different conception rate after artificial insemination and to identify the potential biomarkers. Semen samples were collected from boars which divided into two groups according to the conception rates in the offspring. Seminal plasma metabolites were isolated, purified, and then subjected to Ultra-high Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-qTOF-MS) procession. A total of 576 (Positive ion mode) and 377 (Negative ion mode) metabolites were identified in seminal plasma. Metabolites were identified and categorized according to their major chemical classes, including carboxylic acids and derivatives, organooxygen compounds, amino acids, peptides, and alogues, fatty amides, fatty acyls, benzene and substituted derivatives, purine nucleotides, pyrimidine nucleotides, glycosyl compounds, fatty acids and conjugates. The results showed that 4-Aminobenzoate, Pro-Asn, Ile-Tyr, Homoveratric acid and D-Biotin were higher in semen of boar with higher conception rate (HG) versus lower conception rate (LG) (p < .05), whereas L-Serine, Butoxyacetic acid, S-Methyl-5'-thioadenosine, Capsaicin and 1-O-(cis-9-Octadecenyl)-2-O-acetyl-sn-glycero-3-phosphocholine (PAF) were lower in HG than in LG (p < .05). These metabolites may be considered as candidate biomarkers for different fertility in boars.     

Effect of organic and inorganic selenium supplementation on semen quality and blood enzymes in buffalo bulls

The present study aimed to evaluate the effect of organic and inorganic selenium (Se) supplementation on semen quality and blood serum profiles of buffalo bulls. Nine mature buffalo bulls were divided into three groups: control (non‐supplemented); organic Se (10 mg Sel‐Plex®/head twice weekly) and inorganic Se (10 mg sodium selenite/head twice weekly). Semen was collected twice a week for 3 months during Se supplementation. Semen properties were evaluated from fresh ejaculate. Moreover, fructose concentration, aspartate and alanine transaminase (AST and ALT) activities, total protein and total cholesterol were assayed in seminal plasma. Additionally AST, ALT, testosterone and Se levels were determined in the blood serum. Results showed that Se supplementation significantly (P < 0.05) influences the semen parameters during 3 months of treatment. Organic Se significantly (P < 0.05) increased the percentage of viable sperms compared to inorganic Se and the control group. Fructose concentration was significantly higher (P < 0.05) in the seminal plasma of organic Se‐treated bulls. Serum testosterone and Se concentrations were significantly (P < 0.05) increased in the Se supplemented groups than the control group. In conclusion, Se supplementation improved the parameters of buffalo bull semen and more precisely, organic Se was more effective for the improvement of semen quality and some blood components than inorganic Se.     

Circulating Metabolic Hormones During the Peripubertal Period and Their Association with Testicular Development in Bulls

The objective of the present study was to characterize changes in serum metabolic hormones concentrations from 20 weeks before to 20 weeks post-puberty in bulls and to investigate the associations of metabolic hormones concentrations with testicular development. Leptin concentrations increased from 16 weeks before puberty to 8 weeks post-puberty and insulin concentrations increased from puberty to 8 weeks post-puberty. Growth hormone concentrations decreased after 4 weeks post-puberty, whereas IGF-I concentrations increased from 8 weeks before puberty to 8 weeks post-puberty. During this period, testicular growth was accelerated and testosterone secretion increased substantially, without any significant changes in gonadotropin secretion. Monthly circulating concentrations of leptin, IGF-I and insulin accounted for 63% of the variation in scrotal circumference and 59% of the variation in paired testes volume. In conclusion, the secretion of metabolic hormones was not associated with changes in gonadotropins concentrations. Furthermore, the associations of leptin, IGF-I and insulin concentrations with testes size indicated that these hormones might be involved in a gonadotropin-independent mechanism regulating the testicular development in peripubertal bulls.     

Body growth and plasma concentrations of metabolites and metabolic hormones during the pubertal period in female Shiba goats

It has been shown in various species that the onset of puberty is closely associated with body growth and nutritional state rather than age. The present study was conducted to determine the timing of puberty and to clarify body growth and metabolic changes around the pubertal period in female Shiba goats. Blood samples were collected between 10 to 38 weeks of age from 12 female goats, and plasma concentrations of progesterone, metabolites (glucose, nonesterified fatty acid, ketone body and acetic acid) and metabolic hormones (insulin and insulin-like growth factor-I (IGF-I)) were analyzed. Physical parameters (body weight, withers height and body length) were also measured at the blood sampling. The week when plasma progesterone concentrations first exceeded 1.0 ng/ml was designated as the onset of puberty. The results showed that the average age of the onset of puberty was 27.0 +/- 0.9 (mean +/- SEM) weeks in female Shiba goats. When the goats reached puberty, the average values of body weight and goat body mass index ((body weight (kg)/withers height (cm)/body length (cm)) x 10(3)) were 12.2 +/- 0.5 kg and 5.7 +/- 0.2, respectively. No particular change associated with puberty was apparent for plasma concentrations of the metabolites examined. Plasma insulin concentrations were maintained at lower levels until the onset of puberty, and then they began to gradually increase. Plasma IGF-I concentrations began to gradually increase 1 to 4 weeks before the onset of puberty and this increase continued throughout the peripubertal period. These results imply that IGF-I acts as a peripheral nutritional signal to trigger the onset of puberty in Shiba goats.     

Insulin-like growth factors and IGF-binding proteins in bovine seminal plasma.

Insulin-like growth factor-I (IGF-I) is an important factor for germ cell development and maturation of spermatozoa. Actions of IGFs are modulated by IGF-binding proteins (IGFBPs) that may, depending on their concentration and site of expression, inhibit or enhance effects of IGF-I. We characterized IGFs and IGFBPs in seminal plasma from bulls routinely used for artificial insemination (AI) and from bulls producing poor-quality semen (low mass and individual motility of spermatozoa). IGFs were measured by specific radioimmunoassay in 22 samples of seminal plasma from nine different AI bulls with high (> 76.8%), average (72.8-73.4%), or low (< 69.5%) nonreturn rate (NRR). IGF-I and IGF-II levels were 144 +/- 9 ng/ml (mean +/- SE; range, 79-238 ng/ml) and 144 +/- 10 ng/ml (range, 55-221 ng/ml), respectively, and did not correlate with NRRs. IGF-I concentrations in seminal plasma from bulls producing poor-quality semen (n = 10) were significantly (P < 0.05) greater (194 +/- 26 ng/ml; range, 94-370 ng/ml), whereas IGF-II levels were significantly (P < 0.05) lower (93 +/- 17 ng/ml; range, 38-183 ng/ml) than in AI bulls. Ligand blot analysis of seminal plasma for IGFBPs revealed the presence of a 38-/45-kDa doublet band and a 30-kDa IGFBP. These IGFBPs were identified as IGFBP-3 and IGFBP-5, respectively, by immunoprecipitation using specific antibodies. In addition, a low amount of IGFBP-4 was detected in bovine seminal plasma by immunoprecipitation. There was a marked difference in the activity of IGFBPs between individual bulls, with a relatively small within-bull variance. The differences in IGFBP activities did not correlate with the fertilization capacity of the bulls in vivo or in vitro nor with immunoreactive IGF-I and IGF-II levels in seminal plasma. Our results demonstrate the presence of IGFBPs in bovine seminal plasma. In contrast to human seminal plasma, high activity of IGFBP-3 was detected in seminal plasma of some bulls, suggesting species-specific regulation of IGFBP activity by proteases.     

Relationships of sire scrotal circumference to offspring reproduction and growth

Reproductive and growth data were obtained on 779 and 564 yearling beef heifers and bulls, respectively, that had sires with yearling scrotal circumference data at the San Juan Basin Research Center, Hesperus, CO. Partial regression coefficients of reproductive and growth traits on inbreeding (FXC) and age of the individual and adjusted scrotal circumference of sire (SCSI) were obtained. Growth and reproductive traits of heifers and growth and breeding soundness traits of bulls were analyzed. Separate analyses for each sex were performed, but least squares models were similar. Models included fixed effects of breed, birth year (BY), age of dam (AOD) and the covariates FXC, age (day of birth in heifer analyses) and SCSI. Scrotal circumference of sire was adjusted for age, FXC, AOD and BY using values obtained in a separate analysis. Seminal traits improved as age increased, and there was a seasonal effect present for age of puberty. Inbreeding had a detrimental effect on reproductive traits. Partial regression coefficients for the reproductive traits on SCSI were: age of puberty, -.796 d/cm; age of first calving, -.826 d/cm; julian day of first calving, -.667 d/cm; julian day of second calving, .597 d/cm; most probable producing ability, .132 %/cm; percent sperm motility, -.74 %/cm; percent primary sperm abnormalities, .08 %/cm; percent secondary sperm abnormalities, .92 %/cm; percent normal sperm, -1.28 %/cm; total breeding soundness examination score, .28 units/cm and scrotal circumference, .306 cm/cm. A heritability of .39 was obtained for scrotal circumference.     

Feed restriction in young bulls alters the onset of puberty in relationship with plasma insulin-like growth factor-I (IGF-I) and IGF-binding proteins

The objectives of this study were to evaluate the effect of feed restriction and re-alimentation on the onset of puberty and IGF status in peripubertal male calves and to compare the radioimmunoassay (RIA) and western ligand blotting (WLB) methods for bovine IGFBP-2. Twelve prepubertal 290 d-old Belgian Blue bulls (mean weight: +/- 290 kg) were randomly assigned in three groups: a control group (NG; n = 4) receiving a classic fattening diet to induce "normal" growth (1.48 kg/d), a feed restricted group (RG; n = 4) to obtain reduced growth (0.50 kg/d) and, a severely restricted group (SG; n = 4) to nearly stop growth (0.08 kg/d). The feed restriction period was maintained over a period of 114 d. After the period of differential feeding, all animals received the control feed regime over a period of 100 d. Blood samples were collected at fortnightly intervals. Circulating IGF-I was measured by RIA whereas plasma IGFBPs was evaluated by WLB; IGFBP-2 was additionally quantified by RIA procedure. At the beginning of the trial, IGF-I levels were low (<100 ng/ml) and similar in the three groups in accordance with prepubertal status. In the NG group, a progressive rise in IGF-I was observed from Day 42 to Day 142 whereas in the RG and SG groups, IGF-I levels did not change until the experimental restriction period ended. The delay of the rise in plasma IGF-I was longer for the SG group, IGF-I remained low until 2 wk after the end of the period of restricted feeding. Surprisingly, although differences were detected for IGF-I levels between the three groups, the IGFBP-2 and -3 data, evaluated by WLB could only discriminate between NG and SG group and not between NG and RG. However, by using a RIA method, an IGFBP-2 decrease was observed in the NG group coincident with increasing IGF-I levels. For both RG and SG groups, IGFBP-2 levels remained high throughout the feed restriction period whereas plasma IGFBP-2 levels declined upon feeding in both groups. During this feed restriction period, IGFBP-2 was significantly lower in NG than in RG or SG groups. Moreover, SG group animals had higher levels in plasma IGFBP-2 than RG animals. In conclusion, puberty is characterized by developmental changes in plasma IGF-I and IGFBPs that were altered by feed restriction. Moreover, RIA evaluation of plasma IGFBP-2 is able to better reflect group differences than WLB.     

Effects of supplemental conjugated linoleic acids (CLA) on fresh and post‐thaw sperm quality of Holstein bulls

This study was designed to investigate the effects of feeding‐protected conjugated linoleic acid (CLA) on the semen production and sperm freezability in Holstein bulls. Twelve bulls were randomly assigned to two groups (n = 6 per group). Bulls received the normal diet (control group) or the normal diet top‐dressed with 50 g of CLA (treated group) for 10 weeks. The control group received 40 g/day calcium soap of fatty acid. Fresh and post‐thaw semen quality was assessed on ejaculates collected at the 0, 4, 6, 8 and 10 week of supplementation. Semen evaluations including sperm concentration, motion characteristics (subjective and computer‐assisted), viability (Eosin–Nigrosin), membrane integrity (hypo‐osmotic swelling test) and abnormality were conducted. Semen volume, sperm concentration and total sperm output were not affected by dietary treatment (p > .05). The proportion of spermatozoa with abnormal morphology in fresh semen significantly increased (p < .05) in the CLA‐fed group compared to control group. Also, in CLA‐fed group, the proportion of post‐thaw spermatozoa with abnormal morphology at week 10 of trial was significantly higher in CLA than control group (p < .05). Progressive motility tended to be increased in the CLA‐fed group, although dietary supplementation did not affect other CASA parameters or viability in fresh and frozen‐thawed sperm. In this study, CLA supplementation had little positive effect on fresh or post‐thaw sperm quality of Holstein bulls.     

Interrelationship of growth hormone AluI polymorphism and hyperketonemia with plasma hormones and metabolites in the beginning of lactation in dairy cows

A polymorphic site of the growth hormone gene (AluI polymorphism) that results in an amino acid change at position 127 of the protein chain (leucine, L to valine, V) has been linked to differences in circulating metabolites and metabolic hormones of calves and bulls and to milk yield traits of lactating cows. Our objective was to investigate the interrelationship of this polymorphism with plasma concentrations of β-hydroxybutyrate, insulin, IGF-I and leptin in postpartum dairy cows. Blood samples were taken from clinically healthy, spring-calving, group-fed Holstein-Friesian cows (n = 257; 7 large-scale farms) 4–13 days after calving. Of all herds, 100 cows had plasma β-hydroxybutyrate levels above 1.2 mmol/l and 157 cows were normoketonemic. The proportion of valine carriers and LL cows was not different within groups of normo- and hyperketonemic animals. Genotype was not associated with plasma β-hydroxybutyrate, insulin, IGF-I and leptin levels either in all of the herds or in the two with the highest proportion of the valine allele carriers (n = 28, 72%). We found significantly lower insulin, IGF-I and leptin concentrations in the presence of hyperketonemia compared to normoketonemic cows. There were strong negative correlations between BHB and the other blood parameters, while insulin, IGF-I and leptin were positively related to each other. In conclusion, in the first two weeks after calving we could not demonstrate any effect of AluI polymorphism on plasma concentrations of β-hydroxybutyrate and metabolic hormones studied. Hyperketonemia was associated with a significant decrease in insulin, IGF-I and leptin blood levels. We infer that cows homozygous for the leucine allele or carrying the valine allele may have a similar endocrine and metabolic response to the challenge of increased nutrient demand early postpartum and that the presence of hyperketonemia is mainly linked to the hormonal and metabolic changes occurring at the onset of lactation.     

Effect of nutrition during calfhood and peripubertal period on serum metabolic hormones, gonadotropins and testosterone concentrations, and on sexual development in bulls

The objective of the present study was to characterize the effects of nutrition on circulating concentrations of metabolic hormones, gonadotropins, and testosterone during sexual development in bulls. Nutrition regulated the hypothalamus-pituitary-testes axis through effects on the GnRH pulse generator in the hypothalamus and through direct effects on the testes. Pituitary function (gonadotropin secretion after GnRH challenge) was not affected by nutrition. However, nutrition affected LH pulse frequency and basal LH concentration during the early gonadotropin rise (10-26 weeks of age). There were close temporal associations between changes in insulin-like growth factor-I (IGF-I) concentrations and changes in LH pulse frequency, suggesting a role for IGF-I in regulating the early gonadotropin rise in bulls. The peripubertal increase in testosterone concentration was delayed in bulls with lesser serum IGF-I concentrations (low nutrition), suggesting a role for IGF-I in regulating Leydig cell function. Serum IGF-I concentrations accounted for 72 and 67% of the variation in scrotal circumference and paired-testes volume, respectively (at any given age), indicating that IGF-I may regulate testicular growth. Bulls with a more sustained elevated LH pulse frequency during the early gonadotropin rise (high nutrition) had greater testicular mass at 70 weeks of age relative to the control group (medium nutrition), despite no differences in metabolic hormone concentrations after 26 weeks of age. Therefore, gonadotropin-independent mechanism regulating testicular growth might be dependent on previous gonadotropin milieu.     

Total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs

The aim of this study was to evaluate the total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs. The study was conducted on 10 infertile and 10 fertile dogs of various breeds. Infertility was defined as conception failure at least three matings with different bitches. Semen was collected by manual manipulation. The sperm concentration and motility parameters were evaluated using CASA Hamilton Thorne, Vers. IVOS 12.3. The morphology of spermatozoa and the percentage of live and dead sperm cells were assessed microscopically, total antioxidant capacity and the content of SH‐groups in seminal plasma were determined spectrophotometrically, the contents of protein peroxidation markers in seminal plasma, bityrosine and formylokinurenine, were determined using spectrofluorimetric methods. Sperm concentration and total sperm count were significantly (p < 0.05) lower in infertile dogs than in fertile dogs (99.92 ± 3 0.05 × 10⁶/ml vs. 282.07 ± 48.27 × 10⁶/ml; 214.19 ± 114.74 × 10⁶ vs. 747.57 ± 210.94 × 10⁶, respectively). The percentage of spermatozoa with normal morphology and the most determined motility parameters differed significantly (p < 0.05) between both groups. The mean values of total antioxidant capacity in the seminal plasma were significantly (p < 0.05) lower (19.95 ± 20.94 vs. 25.66 ± 23.18 µmol/g protein), whereas the mean contents of bityrosine and formylokinurenine in seminal plasma were significantly (p < 0.05) higher in infertile dogs than in fertile dogs (3.71 ± 4.83 µg/mg protein vs. 1.55 ± 2.00 µg/mg protein and 0.37 ± 0.45 µg/mg protein vs. 0.14 ± 0.08 µg/mg protein, respectively). In conclusion, the obtained results suggest that the poor semen quality and infertility in dogs could be associated with lowered total antioxidant capacity and increased protein peroxidation in seminal plasma as a consequence of oxidative stress.     

Estimation of endogenous levels of osteopontin,total antioxidant capacity and malondialdehyde in seminal plasma: Application for fertility assessment in buffalo (Bubalus bubalis) bulls

This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high‐fertile (conception rate >50%) and subfertile bulls (conception rate <40%). A total of 100 ejaculates (10 ejaculates from each bull) were collected through artificial vagina method. The concentration of OPN, TAC and catalase (CAT) of high‐fertile bulls was found to be higher (p < .05) than that of subfertile bulls. Further, MDA level in seminal plasma was found to be lower (p < .05) in high‐fertile bulls compared with subfertile bulls. The fertility status had no effect on the superoxide dismutase (SOD) concentration in seminal plasma of both the groups. The levels of OPN (r = .678, p = 0.013) and TAC (r = .648, p = .042) were found to be positively correlated with bull fertility and the level of MDA (r = ?.718, p = .019) was found to be negatively correlated with bull fertility. However, the fertility of bulls was not found to be significantly correlated with SOD, CAT and sperm motility. In conclusion, seminal OPN, TAC and MDA tended to be more realistic in identification of subfertile bulls from breeding herds.     

Effect of vitamins,probiotics and protein level on semen traits and seminal plasma biochemical parameters of post-moult male broiler breeders

1. A study was designed to investigate the comparative effect of supplementary vitamin E, vitamin C, probiotics and dietary crude protein concentration on semen traits and seminal plasma biochemical parameters in male broiler breeders after Zn-induced moulting.

2. A total of 180 male broiler breeders were induced to moult at 65 weeks of age by mixing ZnO in diet at the rate 3000?mg/kg of feed. After moulting, the males were divided into six groups that were given feed supplemented with: vitamin C (500?IU/kg), vitamin E (100?IU/kg), protein (140?g CP/kg), probiotics (50?mg/kg) and the combination of these components, while one group was kept as a control. Semen samples were collected weekly and semen volume, spermatozoa concentration, motility and dead spermatozoa percentage were determined. Seminal plasma was separated to determine the concentration of total antioxidant capacity (TAC), homocysteine, paraoxonase (PON1), arylesterase, ceruloplasmin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities.

3. Semen volume was significantly higher in the vitamin E and C groups compared to the control. Spermatozoa motility was higher in the vitamin E group and dead spermatozoa percentage was significantly lower in the vitamin C group compared with the control group.

4. Seminal plasma TAC was higher in the vitamin E group, homocysteine was lower in the vitamin C and E groups. PON1 was higher in the combination group. Arylesterase increased significantly in the vitamin C and combination groups over time. Seminal plasma AST was significantly lower in the vitamin C and E supplemented groups whereas ALT decreased significantly only in the vitamin E group compared with the control. Higher concentrations of ceruloplasmin were observed in the combination group compared with the other treatments.

5. It was concluded that additional vitamin E and C or their combination was the most potent nutrient treatment for improving the semen traits and seminal plasma biochemical characteristics in male broiler breeders after Zn-induced moulting.     

The effect of feeding whole cottonseed on the fertility of bulls

SUMMARY 23 bulls were fed whole cotton seed (WCS) for 9 months, which resulted in the ingestion of 7.6 to 19.8 g free gossypol per head daily, or 20.4 to 50.8 mg per kg body weight daily. This level of free gossypol ingestion exceeded that of previous studies of a shorter duration in which gossypol was deemed to have affected bull fertility. There was no significant difference between treated and untreated bulls for total spermatozoal abnormalities, secondary spermatozoal abnormalities, scrotal circumference, scrotal circumference per kg body weight, percentage passing the Society for Theriogenology examination for breeding soundness, and mean breeding soundness examination score. There was a higher proportion of primary spermatozoal abnormalities in untreated than in WCS-fed bulls (P = 0.05). Of the bulls that were prepuberal at the onset of the trial (n = 9), the mean age of onset of puberty was not significantly different in the WCS group than in the untreated group, but it approached significance (P = 0.05). There was no significant difference in the scrotal circumference at the onset of puberty between the two groups. Analysis of the bulls' drinking water showed they could have ingested sufficient minerals in the water to bind with the free gossypol ingested, thereby detoxifying it. This may also explain why the results of previous trials have been so variable.     

Cross comparison of seminal plasma proteins from cattle and buffalo (Bubalus bubalis)

The objective of this study was to evaluate seminal plasma proteins from cattle and buffalo (Bubalus bubalis), to identify differences between related species. Sixteen buffaloes and 16 cattle between 30 and 60 months of age were used. Semen collection was performed by electroejaculation, followed by macroscopic and microscopic subjective analyses. After analysis, the samples were centrifuged at 800 g for 10 min, and the supernatant (seminal plasma) was recentrifuged at 10,000 g for 30 min at 4°C. The total protein concentration was determined by the Bradford method, and the proteins were digested in solution for mass spectrometry (nLC-MS/MS). Multivariate statistical analysis was used to evaluate the proteomics results by non-hierarchical clustering the considering exponentially modified protein abundance index (emPAI). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used for clustering. Proteomics identified 78 proteins, and multivariate analysis showed 4 that were over-expressed in buffaloes (cystatin C, prosaposin, peptide YY and keratin type II cytoskeletal 5) and 9 in cattle (spermadhesin-1, seminal plasma protein PDC-109, ribonuclease 4, metalloproteinase inhibitor 2, acrosin inhibitor 1, seminal ribonuclease, C-type natriuretic peptide, angiogenin-1 and osteopontin). Among the proteins identified in seminal plasma, the C-type natriuretic peptide and metalloproteinase inhibitors were described for the first time in buffaloes. Some protease inhibitors were found over-expressed in buffaloes, and important proteins in seminal plasma of cattle were not identified or were found at lower expression levels in buffaloes, which can contribute to reproductive performance in this species.     

Effect of weaning method on body weight,testicular growth,sexual behavior,age of puberty,and testosterone concentration of Beetal bucks

The current study evaluates the effects of early (8th week) and late (16th week of age) weaning of male goat kids on their body growth, testicular growth, sexual behavior, plasma testosterone concentration, and pubertal age. Early (n = 6) and late (n = 7) weaned Beetal bucks were weekly monitored from 18th to 38th week for their body weight, scrotal circumference, testicular volume, testicular echogenicity (via ultrasonography), sexual activities, and plasma testosterone concentration. In comparison to early-weaned, late-weaned bucks showed a marked increase (p < .05) in body weight (11.4 ± 0.8 vs. 13.7 ± 0.6kg), testicular volume (44.1 ± 7.2 vs. 79.8 ± 18.7cm³), scrotal circumference (10.7 ± 0.6 vs. 12.8 ± 0.7cm), and testicular echogenicity (28.3 ± 2.7 vs. 38.3 ± 2.1) from 18th, 28th, 21st, and 24th week onward, respectively. Sexual activities started earlier in late- than early-weaned bucks (22nd vs. 25th week, respectively). Moreover, the sexual behavior index was better (p < .05) after the 34th week in late than early-weaned bucks. The plasma concentration of testosterone (at 39 weeks of age) was relatively more and the onset of puberty was 2–3 weeks earlier (p < .05) in late than early-weaned bucks. In conclusion, age-based early weaning of male kids impairs their testicular growth, sexual behavior, and age at puberty compared to conventional weaning.     

Long‐term Effects of Pyrethrin and Cyfluthrin,a Type II Synthetic Pyrethroid,Insecticide Applications on Bull Reproductive Parameters

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 < 0.01) for sperm with primary defects; bulls in CONT group had a greater (p = 0.01) percentage of sperm with primary defects than bulls treated with insecticides at week 18. Overall and progressive sperm motility, normal sperm morphology, secondary sperm defects and serum testosterone concentrations changed (p < 0.01) over time in all bulls; however, treatment did not affect (p ≥ 0.13) any of these parameters. There were also no treatment effects (p ≥ 0.08) on bull body weight, body condition score or scrotal circumference. The use of pyrethrin‐ and cyfluthrin‐based insecticides, regardless of application, did not negatively affect reproductive parameters in beef bulls when administered over 18 weeks.     

Relationship of age to body weight,scrotal circumference,testicular ultrasonograms,and semen quality in Sahiwal bulls

The objective of the present study was to determine the relationship of age to body weight (BW), scrotal circumference (SC), number of pixels of testicular ultrasonograms (NP), and semen quality in Sahiwal bulls. The study was based on 128 Sahiwal bulls of different age groups (from 0 to >100 months of age). Bulls were evaluated for SC, BW, and NP. Semen was evaluated once a week for five consecutive weeks from regularly collected donor bulls (n = 86) ranging in age from 25–30 to >100 months. Ejaculate volume, sperm motility, sperm concentration, sperm morphology, percent live sperms, sperm plasma membrane integrity, and normal acrosome were compared among different age groups. Mean SC and BW increased (P < 0.05) in a curvilinear manner from birth to >100 months of age. Mean NP of testicular ultrasonograms increased (P < 0.05) from 0 to 24 months and then plateaued until >100 months of age. Body weight, SC, and NP were positively correlated with age from birth until >100 months (r = 0.91, 0.87, and 0.40, respectively). Ejaculate volume (5.7 ± 0.2 vs. 4.6 ± 0.09 ml) and sperm concentration (1,281.6 ± 17.7 vs. 1,115.8 ± 55.9 × 10⁶/ml) increased (P < 0.05) in mature bulls compared to younger ones. However, motility (68.6 ± 0.3%), plasma membrane integrity (50.8 ± 1.0%), and normal acrosome (74.8 ± 0.5%) remained insignificant due to age. In six of eight age groups studied, morphological abnormalities were well within the range (18.1 ± 0.3%). In conclusion, the BW, SC, and NP of testicular ultrasonograms, ejaculate volume, and concentration increased with age. Moreover, semen quality is fairly independent of age except volume and concentration in Sahiwal bulls.     

Effects of supplementation of iodixanol to semen extender on quality and fertilization ability of frozen–thawed Thai native bull sperm

This study investigates the effects of iodixanol supplementation in varied concentrations to Tris egg yolk (TEY) extender on the quality and fertilization ability of frozen–thawed sperm of Thai native bulls. Each ejaculate was divided into four different groups, as follows: sperm were treated with TEY extender (control group) and TEY extender supplemented with three different concentrations of iodixanol (1.25%, 2.50% and 5.00%). Semen straws were frozen in liquid nitrogen vapor. After thawing, sperm motility characteristics, viability, plasma membrane integrity and acrosome integrity were determined. Also, frozen–thawed spermatozoa from all groups were used for in vitro fertilization and artificial insemination (AI) in natural estrus Thai native cows. The results showed that the post‐thaw quality of the 2.50% iodixanol group was superior to the other iodixanol groups (P < 0.05). However, iodixanol had no beneficial effect on post‐thaw sperm in vitro fertilization ability and pregnancy rate after AI (P > 0.05). It can be concluded that the supplementation of 2.50% iodixanol extender significantly improves the progressive motility, viability, plasma membrane integrity and acrosome integrity of cryopreserved semen from Thai native bulls, but it has no beneficial effect on in vitro fertilization ability and pregnancy rate after AI.