Effects of age and environmental factors on semen production and semen quality of Austrian Simmental bulls

More than 90% of the breeding stock of Austrian dual purpose Simmental cows is artificially inseminated. Knowledge of factors affecting sperm production and semen quality is of importance with regard to reproductive efficiency and thus genetic improvement as well as for the productivity and profitability of AI centers. Hence, semen data from two Austrian AI centres collected in the years 2000 and 2001 were evaluated. In total, 3625 and 3654 ejaculates from 147 and 127 AI bulls, respectively, were analysed regarding ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. Effects accounted for were the bull (random), age of bull, collection interval, number of collection on collection day, bull handler, semen collector, temperature on day of semen collection, in the course of epididymal maturation (average temperature of days 1-11 before collection) and during spermatogenesis (average temperature of days 12-65 before collection). Age of bull significantly affected all traits (P<0.01 to P<0.001) except motility score in center 2. Ejaculate volume and total number of spermatozoa increased with age of bull while sperm concentration was lower in higher age classes (center 1). The collection team was also found to significantly influence semen quality traits. With increasing collection interval ejaculate volume and total number of spermatozoa increased significantly (P<0.05 to P<0.001) while collection intervals between 4-9 days and 1-6 days were superior with regard to sperm concentration and percentage of viable spermatozoa, respectively (P<0.10 to P<0.001). First ejaculates were superior with respect to ejaculate volumes, sperm concentrations and total number of spermatozoa per ejaculate (P<0.001). Temperature, either on day of semen collection or during epididymal maturation or spermatogenesis, had important but inconsistent effects on semen production and sperm quality. Overall, however, ambient temperatures in the range of 5-15 degrees C were found to be optimal for semen production.     

Spermatogenesis, sperm output and seminal quality of Holstein bulls electroejaculated after administration of oxytocin

The 12- to 24-month-old Holstein bulls were electroejaculated twice on each of 3 days per week throughout the study. After a 2-week stabilization period and subsequent 2-week pre-treatment period, 7 bulls were given 50 i.u. oxytocin via the jugular vein 10 min before each first ejaculate for 10 weeks. The 7 control bulls were handled identically but did not receive oxytocin. All bulls were castrated at the end of the study. Oxytocin was without effect on spermatogenesis (P greater than 0.10). Oxytocin did not alter the total number of spermatozoa harvested per collection day (P greater than 0.10), but increased the number of spermatozoa in first ejaculates by an average of 34.2% (P less than 0.025). Oxytocin did not affect sperm quality (P greater than 0.10) as judged by the motility of spermatozoa in fresh semen or by the motility or percentage of spermatozoa with intact acrosomes in thawed semen. It is concluded that 50 i.u. oxytocin enhanced sperm output in first ejaculates of electroejaculated bulls without altering daily sperm production or seminal quality.     

Effect of semen collection practices on sperm characteristics before and after storage and on fertility of stallions

This study analyzed effects of different methods and intervals of semen collection on the quantity and quality of fresh, cool-stored, and frozen-thawed sperm and fertility of AI stallions. In Experiment 1, ejaculates were obtained from six stallions (72 ejaculates per stallion) using fractionated versus non-fractionated semen collection techniques. Initial sperm quality of the first three jets of the ejaculate was not different from that of total ejaculates. Centrifugation of sperm-rich fractions before freezing improved post-thaw motility and sperm membrane integrity when compared to non-centrifuged sperm-rich fractions or non-fractionated centrifuged ejaculates (P<0.05). In Experiment 2, semen from four stallions (60-70 ejaculates per stallion) was collected either once daily or two times 1h apart every 48 h. The first ejaculates of double collections had significantly higher sperm concentrations, percentages of progressively motile sperm (PMS) after storage for 24h at 5 degrees C and lower percentages of midpiece alterations than single daily ejaculates. Semen collected once daily showed significantly lower values of live sperm after freezing and thawing than the first ejaculate of two ejaculates collected 1h apart every 48 h. In Experiment 3, semen was collected from 36 stallions (> or =12 ejaculates per stallion) during the non-breeding season and the time to ejaculation and the number of mounts was recorded. When time to ejaculation and the number of mounts increased, volume and total sperm count (TSC) also increased (P<0.05), whereas a decrease was observed in sperm concentration, percentage of PMS after storage for 24 h at 5 degrees C, percentage of membrane-intact sperm in fresh semen (P<0.05) as well as motility and percentage of membrane-intact sperm of frozen-thawed sperm (P<0.05). In Experiment 4, AI data of 71 stallions were retrospectively analyzed for the effect of number of mounts per ejaculation and frequency, time interval of semen collections on pregnancy, and foaling rates (FRs) of mares. Semen volume increased, but sperm concentration and percentage of PMS after 24-h cool-storage decreased with increasing number of mounts on the phantom (P<0.05). A statistically significant inter-relationship was demonstrated between frequency and interval of semen collection and FR. Mares inseminated with stallions from which semen was collected frequently (> or =1 on an average per day) showed significantly higher FRs than mares inseminated with semen from stallions with a daily collection frequency of 0.5-1 or <0.5. FR of mares inseminated with stallions having 0.5-1 days between semen collections was significantly better than FR of mares that were inseminated with stallions having semen collection intervals of 1-1.5 days or >2.5 days.     

Associations between insulin-like factor 3, scrotal circumference and semen characteristics in young Norwegian Red bulls

With the integration of genomic selection in the cattle artificial insemination (AI) industry, bulls are selected for their semen production capacity and fertility at a younger age than previously. Norwegian Red bull calves selected as candidates to become future AI bulls based on their genomic breeding value are kept in a performance testing station from around the age of 3–12 months, allowing for sample collection and analysis of different parameters during their pre- and peripubertal period. Insulin-like factor 3 (INSL3) is a small peptide hormone specifically secreted by the mature Leydig cells of the testes. In the foetus, it induces the first phase of testicular descent and is considered to reflect Leydig cell development during puberty; it could therefore be an interesting early indicator of future semen production capacity. The main objective of our study was to evaluate the relationship between INSL3, scrotal circumference (SC), and semen characteristics. This is the first time INSL3 was measured in the Norwegian Red population. We collected blood samples for analysis of INSL3 from 142 Norwegian Red bulls at the performance testing station and measured their SC on the same day. Altogether, measurements were made at four time points: upon arrival at the performance testing station (quarantine (Q): 2–5 months) and later at approximately 6, 9 and 12 months of age. Information on season and place of birth were made available from the database of the breeding company Geno, together with data on semen characteristics from the test station and the AI station. The median SCs for age groups Q, 6, 9, and 12 were 15, 21.5, 29, and 34 cm, respectively. INSL3 was shown to be positively correlated with SC (R = 0.4) but not with any of the semen characteristics. Similarly, we found no correlation between SC and sperm characteristics from data on ejaculates analysed at the performance testing station and AI station. The mean sperm volume for the 31 selected bulls with at least 10 ejaculates produced in the AI station increased from 2.3 ml at the performance testing station to 6.4 ml at the AI station. The corresponding increase in mean sperm concentration was from 497 million/ml to 1 049 million/ml. We conclude that INSL3 exhibits high inter-individual variability in the Norwegian Red bull population, which cannot be explained by the parameters measured in this study. At present, INSL3 cannot be used as a biomarker of sperm production in this breed.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

Relationship between semen quality and pixel-intensity of testicular ultrasonograms after scrotal insulation in beef bulls

The objective of this study was to determine the relationship between semen quality and testicular pixel-intensity derived from image analysis of ultrasonograms after scrotal insulation in bulls. In addition, the ability to predict semen quality based on testicular pixel-intensity was evaluated. Sixteen beef bulls were selected on the basis of satisfactory semen quality and normal testicular ultrasonogram appearance. Bulls were allocated into two groups for scrotal insulation for 4 days (group 1) or 8 days (group 2). Semen was collected and evaluated twice weekly and testicular ultrasonograms were evaluated once weekly for 8 weeks after removal of scrotal insulation. In general, the percentages of motile and morphologically normal spermatozoa decreased below pre-insulation levels from 1 to 5 weeks after scrotal insulation removal. Overall, group 1 had greater (P < 0.01) percentages of motile and normal spermatozoa than group 2. Mean testicular pixel-intensity (PI), and the number of pixels corresponding to the intensity that occurs most frequently (NP) decreased in the first 2-3 weeks after scrotal insulation, coincidently with the decrease in sperm motility and normal morphology. When the entire data set was evaluated, there was no association between testicular PI or NP with semen quality observed at the same week of ultrasound examinations. However, regression models indicated that testicular PI and NP accounted for 13-25% of the variation in sperm motility and morphology in ejaculates collected 2-4 weeks after ultrasound exam. Testicular PI and NP had moderate sensitivity and negative predictive values (64.5-82.6%), but low specificity and positive predictive values (33.3-61.2%) as predictors of satisfactory semen quality (> or = 60% motile spermatozoa and > or = 70% morphologically normal spermatozoa) for ejaculates collected 2-4 weeks after ultrasound exam. In conclusion, the deleterious effects of scrotal insulation on semen quality were dependent on the length of the period of insulation and were associated with changes in testicular ultrasonogram pixel-intensity. Testicular ultrasonogram pixel-intensity had a better association with future semen quality than with present semen quality and was a better predictor of unsatisfactory semen quality than satisfactory semen quality.     

The importance of porcine sperm parameters on fertility in vivo

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.     

French field results (1985-2005) on factors affecting fertility of frozen stallion semen

Results on procedures for freezing stallion semen and the subsequent fertility during 20 years are presented. The present system applied in French National Stud includes: (1) a freezing protocol (dilution in milk, centrifugation and addition of freezing extender (INRA82+egg yolk (2%, v/v)+glycerol (2.5%, v/v) at 22 degrees C, a moderate cooling rate to 4 degrees C and freezing at -60 degrees C/min in 0.5-ml straws); (2) selection of ejaculates showing post-thaw rapid motility >35%; and (3) an insemination protocol (mares examined once daily, two AI of 400 x 10(6) spermatozoa 24 h apart before ovulation, sufficient number of straws to have the possibility to perform six AI of 400 x 10(6) total spermatozoa, i.e. 2.4 x 10(9) total spermatozoa available per mare per season). This system was applied to >110 stallions per year, the average post-thaw motility of ejaculates was 50% (>1800 ejaculates) before selection. The semen freezability was defined as the number of selected ejaculates divided by the total number of ejaculates frozen. Of the stallions, 5, 4, 5, 21 and 64% had semen freezability of 0-10, 10-33, 33-60, 60-90 and over 90%, respectively. Per-cycle pregnancy rate was 45-48% (>1500 mares per year, 1.8 cycles per mare) and foaling rate 64%. In comparison, per-cycle pregnancy rate and foaling rate of mares hand-mated to stallions were 57-59% and 64%, respectively. The average number of straws used was 32-35 (1.75 x 10(9) total spermatozoa) per mare per season. According to our results and the literature, the most important factors for improving fertility of frozen equine semen include: (1) a low concentration of glycerol (2-3.5% final concentration); (2) a suitable base extender for freezing like Lactose-Glucose EDTA or INRA82; (3) a post-thaw motility >30-35%; and (4) a sufficient number of spermatozoa per mare per season (1.5-2 x 10(9) total spermatozoa for two to three cycles) divided into small units. Numbers of spermatozoa, lower than 750.10(6) total spermatozoa per cycle, could result in lower per-cycle pregnancy rate with higher additional costs for management of mares. Because there are no particular regulations on quality and quantity of equine semen in the European Community, there is a need for the uniformity of information about frozen semen. A codification is suggested, based on the number of spermatozoa available per mare per season, the post-thaw motility and the final glycerol concentration.     

Effect of age and genetic group on characteristics of the scrotum,testes and testicular vascular cones,and on sperm production and semen quality in AI bulls in Brazil

The objectives were to determine the effects of age and genetic group on characteristics of the scrotum, testes and testicular vascular cones (TVC), and on sperm production and semen quality in 107 Bos indicus, B. taurus and cross-bred bulls at three artificial insemination (AI) centers in Brazil. In addition, predictors of sperm production and semen quality were identified. In general, scrotal circumference (SC), scrotal shape score, scrotal neck perimeter, and testicular size (length, width and volume) increased (P < 0.05) with age. Although there were no significant differences among genetic groups for SC or testicular size, B. indicus bulls had the least pendulous scrotal shape, the shortest scrotal neck length, and the greatest scrotal neck perimeter (P < 0.05). Fat covering the TVC was thinner (P < 0.05) in bulls < or = 36 months of age and in B. taurus bulls than in older bulls and B. indicus bulls, respectively. Age and genetic group did not affect testicular ultrasonic echotexture. B. indicus bulls tended (P < 0.1) to have the lowest average scrotal surface temperature (SST). In general, ejaculate volume, total number of spermatozoa and number of viable spermatozoa increased (P < 0.05) with age. However, there was no significant effect of age on sperm concentration, motility, major and total defects. The proportion of spermatozoa with minor defects was highest (P < 0.05) in bulls 37-60 months of age. B. indicus bulls had higher (P < 0.01) sperm concentration, total number of spermatozoa and number of viable spermatozoa than B. taurus bulls, with intermediate values for cross-bred bulls. Increased sperm production was associated with increased testicular volume, SC, TVC fat cover, and SST top-to-bottom gradient. Decreased semen quality was associated with increased SC and bottom SST, and decreased scrotal shape, scrotal neck perimeter and vascular cone diameter. In summary, age and genetic group affected the characteristics of the scrotum, testes, and TVC, sperm production and semen quality. In addition, characteristics of the scrotum, testes and TVC were associated with sperm production and semen quality in bulls and could be assessed for breeding soundness evaluation.     

Computerized and Subjective Assessments of Post-thaw Motility of Semen from Finnish Ayrshire AI Bulls in Relation to Non-return Rates

A semen analyser (Lazymot), was used to evaluate post-thaw motilities in 296 batches of semen from 74 Ayrshire bulls used for artificial insemination (AI). Motility was also assessed subjectively. A significant correlation was observed between assessments of motility using the Lazymot analyser and the subjective assessments. There was no correlation between post-thaw motility assessments and non-return rates in relation to the batches examined, which met Finnish criteria for use in AI. This suggests that criteria for post-thaw semen motility should not be increased beyond the present requirement for 40% motile spermatozoa.     

Breed differences in competitive indices of Holstein and Jersey bulls and their association with sperm DNA fragmentation index and plasma membrane integrity

The objective was to evaluate the relationship of a competitive index (CI) determined by heterospermic performance and post-thaw semen quality of the same stored ejaculates. Semen from multiple ejaculates collected in succession from each bull (four Holstein and four Jersey) was pooled. Heterospermic doses (20x10(6)/straw) were made to obtain all possible Holstein-Jersey combinations (16 two-bull combinations) and contained 20x10(6) sperm/mL/bull. Cows at two University dairy farms were inseminated on observed or synchronized estrus. The sire of calves (N=460) were determined and a CI was determined for each bull (based on the number of calves sired). Prior to preparation of the heterospermic doses, a sub-sample of semen from each bull was taken, processed, frozen, and stored concurrently with heterospermic samples. Post-thaw semen samples (homospermic) from each bull were assessed for: sperm morphology, acrosome integrity, sperm motility parameters assessed by computer assisted sperm analysis (CASA), flow cytometry analysis of DNA Fragmentation Index (DFI), and Plasma Membrane Integrity (PMI). Heterospermic performance of Holstein bulls was superior to that of Jersey bulls. The DFI was negatively correlated to CI (r=-0.87; P<0.001), whereas the PMI (r=0.87; P<0.001) and total progressive motility (r=0.74; P<0.05) assessed by CASA were positively correlated to CI. In multivariate regression models, the DFI and PMI accounted for 87% variance in competitive index. In conclusion, bulls with less DFI and higher PMI had higher probabilities of siring calves.     

Prediction of fertility of bovine semen: Preliminary studies with the hamster egg penetration test

The ability of liposome-treated fresh and frozen spermatozoa from two bulls to interact with zona-free hamster oocytes was examined to show whether the in vitro test results would correspond with in vivo fertility as indicated by the 60 to 90 d nonreturn to service rates which, using frozen semen, were 77 and 59%, respectively. The motility of spermatozoa in washed suspensions was also rated. Hamster test results were obtained using three ejaculates from each bull both as fresh and frozen semen. The results with frozen semen corresponded with fertility. The averages of three hamster tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte comparing spermatozoa from the bull with the higher fertility with spermatozoa from the bull with the lower fertility were 91% and 2.7 versus 56% and 1.4, respectively. Spermatozoa washed from frozen semen from the bull with the higher fertility interacted with hamster oocytes at the higher rate even when sperm motility was rated the same for both bulls. By contrast, fresh spermatozoa from the lower fertility bull interacted with hamster oocytes at a higher rate than spermatozoa from the higher fertility bull in six tests, comparing six ejaculates of fresh semen from both bulls. Comparing the higher fertility bull with the lower fertility bull, the average of six tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte were 60% and 1.6 versus 89% and 3.0, respectively. This suggests that this hamster test cannot be used with fresh semen to predict relative levels of fertility of frozen semen. Also, the subjective rating of sperm motility did not correspond with the in vitro oocyte penetrating ability of the spermatozoa.     

Effect of season on fertility of frozen buffalo semen

Twenty double ejaculates from each of ten water-buffalo bulls were collected in June (non-breeding season) and again in November (breeding season). Fresh semen was screened for sperm quantity, motility, eosin uptake, and sperm morphology and was frozen using lactose, skim-milk, and Tris extenders. Thawed semen was checked for motility and Sephadex filtration. Half of each semen batch was used for artificial insemination in the breeding season and the other half during the non-breeding season.Laboratory screening revealed that June semen had a significantly lower Sephadex filtration rate and a higher percentage of abnormal sperm cells, and three June ejaculates were excluded from further processing due to poor sperm motility. In the remaining ejaculates the motility before freezing and the sperm cell quantity were higher in June semen than in November semen. Eosin uptake, mass motility, and post-freeze-motility did not vary with season. November semen produced significantly higher pregnancy rates than June semen over a total of 3220 inseminations in both seasons. Forty percent of the observed seasonality of buffalo fertility was attributable to the male. No fertility differences appeared between extenders used. When November semen was used, the fertility in adult buffaloes in both seasons was higher than in heifers.     

Effects of environmental factors, age and genotype on sperm production and semen quality in Bos indicus and Bos taurus AI bulls in Brazil

The effects of ambient temperature and humidity, month, age and genotype on sperm production and semen quality in AI bulls in Brazil were evaluated. Data from two consecutive years were analyzed separately. Seven Bos indicus and 11 Bos taurus bulls from one artificial insemination (AI) center were evaluated in Year 1 and 24 B. indicus and 16 B. taurus bulls from three AI centers were evaluated in Year 2. Ambient temperature and humidity did not significantly affect sperm production and semen quality, probably because there was little variation in these variables. Month accounted for less than 2% of the variation in sperm production and semen quality. Increased bull age was associated with decreased sperm motility (P<0.10) and increased minor sperm defects (P<0.001) in Year 1. B. indicus bulls had greater (P<0.005) sperm concentration than B. taurus bulls in both years (1.7×10⁹/ml versus 1.2×10⁹/ml in Year 1 and 1.6×10⁹/ml versus 1.2×10⁹/ml in Year 2, respectively). Ejaculate volume was not significantly affected by genotype in Year 1 (6.6 ml versus 6.9 ml in B. indicus and B. taurus bulls, respectively), but B. indicus bulls had greater (P<0.05) total (11.4×10⁹ versus 8.2×10⁹) and viable (6.7×10⁹ versus 4.9×10⁹) numbers of spermatozoa in the ejaculate than B. taurus bulls. In Year 2, B. taurus bulls had greater (P<0.05) ejaculate volume than B. indicus bulls (8.2 ml versus 6.7 ml, respectively) and total and viable number of spermatozoa in the ejaculate were not significantly different between genotypes (10.3×10⁹ versus 9.1×10⁹ and 6.1×10⁹ versus 5.4×10⁹ in B. indicus and B. taurus bulls, respectively). Sperm motility was not significantly affected by genotype (mean, 59%). In Year 1, B. indicus bulls tended (P<0.10) to have more major sperm defects and had more (P<0.05) total sperm defects than B. taurus bulls (11.8% versus 8.7% and 13.6% versus 10.0%, respectively). In Year 2, B. indicus bulls tended (P<0.10) to have more total sperm defects than B. taurus bulls (16.2% versus 13.3%, respectively). In conclusion, neither ambient temperature and humidity nor month (season) significantly affected sperm production and semen quality. B. indicus bulls had significantly greater sperm concentration and B. taurus bulls had significantly fewer morphologically defective spermatozoa.     

Sperm chromatin structure assay of bulls qualified for artificial insemination

The goal of our study was to find the relationship between fertility of bulls qualified for AI and the percentage of spermatozoa with abnormal chromatin structure as an independent parameter. We used the frozen semen of 8 mature bulls from one AI center. Each bull was represented by 3 ejaculates collected with at least 2-week intervals. Bull fertility was calculated on the basis of non-return ratio and was expressed as a scale where 100 points represented the average fertility of all the AI center's bulls. Bulls with lower or higher fertility received a lower or higher score respectively. Fertility scores of bulls used in the study ranged from 83 to 104 . Semen was processed according to the SCSA (sperm chromatin structure assay) method and was analyzed by flow cytometry. "Artificial" alpha(t) (alpha(t)=red/green+red fluorescence) and red fluorescence histograms were used for calculation of COMPalpha(t), SDalpha(t), %Red, %PeakR and MeanR parameters. The percentage of spermatozoa with abnormal chromatin ranged from 1.2% to 23.8%. A large variation among ejaculates was found for bulls with lower fertility. Fertility correlated significantly with COMPalpha(t) (-0.50, P < 0.05), SDalpha(t) (-0.55, P < 0.01), %Red (-0.53, P < 0.01), %PeakR (-0.58, P < 0.01) and MeanR (-0.45, P < 0.05). The SCSA method has a practical application in analyzing spermatogenesis disorders in bulls. If regularly applied, it allows us to identify and eliminate ejaculates with a high level of sperm chromatin abnormalities.     

Estimates of non-genetic effects and genetic parameters for semen traits in Piemontese bulls

Male reproductive performances are often ignored in cattle breeding programmes, although semen traits might be used to improve bull breeding soundness. Effects of genetic and environmental factors on semen production and quality traits were estimated in 693 Piemontese bulls with the aim of providing the first estimates of genetic parameters for semen traits for this breed. Volume and concentrations of individual ejaculates (up to three per each test-day), and volume, concentration, total number of spermatozoa and post-thawing progressive motility of within test-day pooled semen were available for 19 060 ejaculates. Bulls reached the maximum amount of daily semen production after their third year of age, with concentration rapidly increasing until 23 months of age, and then slowly decreasing. Semen volume was at its highest when collection days were at least 15 days apart, whereas the maximum concentration was reached when the interval was 6 days. Heritability estimates were generally moderate (0.14–0.26), and low for progressive motility (0.08). Estimates of genetic correlation among the volumes of the individual ejaculates were high and positive (≥0.79), as were the genetic correlations among their concentrations (≥0.46). Genetic correlations among volume and concentration traits varied from −0.47 (with a 95% high posterior density interval ranging from −0.65 to −0.23) to −0.32 (with a 95% high posterior density interval ranging from −0.55 to −0.09). Progressive motility was unrelated with the other traits, but moderately positively correlated with volumes of the second and third ejaculates. The magnitude of heritabilities showed that selection for semen traits is possible. However, the unfavourable relationship between volume and concentration must be taken into account if a future selection programme is to be established.     

Effect of different levels and sources of zinc supplementation on quantitative and qualitative semen attributes and serum testosterone level in crossbred cattle (Bos indicus x Bos taurus) bulls

An experiment was conducted on 16 crossbred bulls (about 2 years of age, 316.2+/-0.77 kg average body weight), divided into groups I, II, III and IV to study the effect of different levels of Zn supplementation from inorganic and organic sources on semen quality. The animals in the first 3 groups were supplemented with 0, 35 and 70 ppm Zn from Zn sulfate, respectively and the animals in-group IV were supplemented with 35 ppm Zn as Zn propionate. Semen collection and evaluation was done in the first month (to assess semen quality at the start of the experiment) and 7th, 8th and 9th month of experimental feeding to evaluate the effect of supplemental Zn on semen attributes. We gave 6 months for Zn feeding, so that 3 sperm cycles of spermatogenesis had passed and the collected semen reflected the complete effect of Zn supplementation. Six ejaculates from each bull were collected and evaluated for semen quantitative (ejaculate volume, sperm concentration and sperm number per ejaculate) and qualitative characteristics (semen pH, mass motility, individual motility, sperm livability percent and abnormal sperm percent, percent intact acrosome, bovine cervical mucus penetration test, hypo-osmotic sperm swelling test) and activity of seminal plasma enzymes i.e., alkaline phosphatase, acid phosphatase, GOT and GPT. Testosterone level in the blood serum of crossbred bulls was also estimated. Mean values of semen quantitative and qualitative characteristics at the start of the experiment were statistically non significant (P > 0.05) in all the crossbred cattle bulls, however, there were statistically significant differences among the bulls of different groups after 6 months of zinc supplementation. Mean ejaculate volume (mL) was 2.37, 4.70, 5.86 and 6.38, respectively in groups I to IV, indicating a statistically significant (P < 0.05) higher semen volume in Zn-supplemented groups as compared to the control group of bulls. Similarly, sperm concentration (million.mL(-1)), live sperm (%) and motility (%) were significantly (P < 0.01) higher in Zn-supplemented groups as compared to the control group. The results of BCMPT and HOSST revealed a significant improvement in sperm functional ability in all the groups supplemented with Zn as compared to the control group. The activity of alkaline and acid phosphatase in seminal plasma was significantly (P < 0.05) higher in the Zn-supplemented groups, whereas GOT and GPT activities in seminal plasma were significantly (P < 0.05) lower in the Zn propionate supplemented group as compared to the control group. Testosterone concentration (ng.mL(-1)) in blood serum was significantly higher in animals of groups III and IV, as compared to control group. It may be concluded that Zn supplementation either in the inorganic or organic form in the diet of crossbred bulls improved the qualitative and quantitative attributes of semen; however, the number of sperm per ejaculate, mass motility and semen fertility test like bovine cervical mucus penetration was significantly higher in bulls given Zn in an organic form (Zn propionate) as compared to an inorganic form (Zn sulfate).     

Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden

Use of bull semen with high levels of sperm abnormalities, reflecting genital dysfunction, is not recommended for artificial insemination (AI) since it would most likely lead to subfertility. Sperm quality, including sperm morphology, may deteriorate with increasing age of the bull thus becoming a source of concern when using older, progeny-tested AI bull sires. Although a relationship between sperm morphology and fertility after AI in progeny-tested bull sires has been reported, it is yet unclear which sperm abnormalities are most critical. This constituted the core aim of a 22-month long retrospective study in proven (aged 60-84 months at the start of the study) AI sires of the Swedish Red (SR, n=8) and Swedish Holstein (SLB, n=4) breeds where their semen (107 freezing batches in total, built by a single ejaculate (n=3) or pooling two consecutive ejaculates (n=104) collected at 1-3 months interval), were subjected to detailed morphological examinations on wet- and dry, stained smears. Attention was paid to between- and within-bull variations with regard to presence and level of sperm abnormalities. Sperm morphology differed significantly between sires and ejaculates, with 6/12 sires having ejaculates containing >10% of morphologically deviating sperm head shapes, a commonly used threshold for young AI bulls in Sweden. However, with the exception of pear-shaped or narrow-at-the-base anomalies, the mean values for individual defects were always within the limits expected for a normal bull sire, and were therefore considered acceptable. The percentage of morphologically normal spermatozoa was positively related to fertility, whose output differed significantly among bulls. Among sperm abnormalities, the proportion of morphologically deviating sperm head shapes were negatively correlated with fertility, pear-shaped sperm heads in particular. In conclusion, the relationship between sperm morphology and fertility after AI calls for frequent (2-3 months interval) detailed assessments of sperm morphology in AI stud bull sires.     

A Nonsense Mutation in TMEM95 Encoding a Nondescript Transmembrane Protein Causes Idiopathic Male Subfertility in Cattle

Genetic variants underlying reduced male reproductive performance have been identified in humans and model organisms, most of them compromising semen quality. Occasionally, male fertility is severely compromised although semen analysis remains without any apparent pathological findings (i.e., idiopathic subfertility). Artificial insemination (AI) in most cattle populations requires close examination of all ejaculates before insemination. Although anomalous ejaculates are rejected, insemination success varies considerably among AI bulls. In an attempt to identify genetic causes of such variation, we undertook a genome-wide association study (GWAS). Imputed genotypes of 652,856 SNPs were available for 7962 AI bulls of the Fleckvieh (FV) population. Male reproductive ability (MRA) was assessed based on 15.3 million artificial inseminations. The GWAS uncovered a strong association signal on bovine chromosome 19 (P = 4.08×10⁻⁵⁹). Subsequent autozygosity mapping revealed a common 1386 kb segment of extended homozygosity in 40 bulls with exceptionally poor reproductive performance. Only 1.7% of 35,671 inseminations with semen samples of those bulls were successful. None of the bulls with normal reproductive performance was homozygous, indicating recessive inheritance. Exploiting whole-genome re-sequencing data of 43 animals revealed a candidate causal nonsense mutation (rs378652941, c.483C>A, p.Cys161X) in the transmembrane protein 95 encoding gene TMEM95 which was subsequently validated in 1990 AI bulls. Immunohistochemical investigations evidenced that TMEM95 is located at the surface of spermatozoa of fertile animals whereas it is absent in spermatozoa of subfertile animals. These findings imply that integrity of TMEM95 is required for an undisturbed fertilisation. Our results demonstrate that deficiency of TMEM95 severely compromises male reproductive performance in cattle and reveal for the first time a phenotypic effect associated with genomic variation in TMEM95.