Oligo-dT selected spermatozoal transcript profiles differ among higher and lower fertility dairy sires.

Spermatozoal messenger RNA (mRNA) has the potential as a molecular marker for sire fertility because this population can reflect gene expression that occurred during spermatogenesis and may have a functional role in early embryonic development. The goal of this study was to compare the oligo-dT selected spermatozoal transcript profiles of higher fertility (Conception Rate (CR) 1.8-3.5) and lower fertility (CR -2.9 to -0.4) sires using Ribonucleic Acid Sequencing (RNA-Seq). A total of 3227 transcripts and 5366 transcripts were identified in the higher and lower fertility populations, respectively. While common transcripts between the two populations were identified (2422 transcripts), several transcripts were also unique to the fertility populations including 805 transcripts that were unique to the higher fertility population and 2944 transcripts that were unique to the lower fertility population. From gene ontological analysis, the transcripts unique to each fertility population differed in Biological Processes (BP), including enrichment of regulatory transcripts for growth and protein kinase activity in the higher fertility bulls. Biological variation in transcript presence among individual sires was also found. Of the candidate fertility spermatozoal transcripts chosen from the RNA-Seq population analysis reported here and previous publications, COX7C was negatively correlated with sire fertility. Using high-throughput sequencing, candidate spermatozoal transcripts were identified for further study as potential markers for sire fertility.

Effects of airport screening X-irradiation on bovine sperm chromatin integrity and embryo development.

Biological samples, including cryopreserved sperm, are routinely X-rayed during air shipment. The goal was to investigate the impact of X-irradiation used for checked and carry-on luggage on bovine sperm chromatin integrity and postfertilization in vitro embryonic development. Frozen domestic bull sperm (Bos taurus) (n=9 bulls) stored in a dry shipper (-160 degrees C) was screened by X-irradiation 0, 1, 2, and 3 times as either carry-on or checked luggage. Duplicate straws were thawed, and sperm were assessed for chromatin damage using the sperm chromatin structure assay (SCSA) and by postfertilization in vitro developmental competence of mature oocytes. Multiple exposure to X-rays did not significantly affect sperm chromatin integrity assessed by SCSA. There were lower proportions of oocytes cleaved (P=0.07; 21.6+/-3.1% vs. 29.4+/-3.1%, 24.9+/-3.1%, and 25.7+/-3.3% for 3 vs. 0, 1, and 2 times, respectively; least-squares means+/-SEM) and that developed to blastocysts (P=0.06; 9.0+/-1.7% vs. 13.8+/-1.7%, 11.5+/-1.7%, and 12.6+/-1.9%, respectively) when fertilization was performed with sperm X-rayed 3 times using checked luggage irradiation; developmental competence (percentage cleaved embryos becoming blastocysts) was unaffected. There were no deleterious effects of other X-irradiation treatments on embryo development. We inferred that screening by X-irradiation may reduce the ability of sperm to activate oocyte cleavage after multiple exposures at the checked luggage dose. However, there was no evidence that competence of embryos to become blastocysts was reduced by X-irradiation (45.4+/-5.7%, 40.4+/-5.7%, 46.4+/-6.1%, and 41.8+/-5.7% for 0, 1, 2, and 3 doses, respectively), but potential long-term epigenetic effects are unknown.

Bovine viral diarrhea virus is inactivated when whole milk from persistently infected cows is heated to prepare semen extender.

Bovine viral diarrhea virus (BVDV) can be present in cryopreserved bovine semen and be transmitted through artificial insemination. Because BVDV can be shed in milk, the virus might also be introduced as a contaminant of milk-based semen extenders. Thus, the purpose of this study was to evaluate the epidemiologic risk of using heated, BVDV-contaminated milk to prepare semen extender. Milk was obtained from cows free of and persistently infected (PI) with BVDV. Six replicates of milk samples were processed by heating (85-92.2 degrees C, 10min). Samples of milk collected before and after heating were assayed for BVDV. Additionally, milk was injected intravenously into eight BVDV seronegative calves to monitor for seroconversion and viral infection. Virus was not detected in any milk samples from negative animals. Virus was consistently isolated from unheated milk samples from PI cows by passage of somatic cells, ultracentrifugation, and animal inoculation. Virus was usually detected in these samples by RT-nPCR (reverse transcription nested polymerase chain reaction). In heated milk samples from PI cows, no infectious BVDV was detected using any technique, but viral RNA was detected using RT-nPCR in four of six replicates. Bovine viral diarrhea virus in milk from PI cows was inactivated by heating. Therefore, properly heated milk used in semen extenders will not result in transmission of infectious BVDV. Although RT-nPCR detected the presence of viral RNA in milk samples after heating, the virus was not infectious as demonstrated by lack of replication despite using multiple sensitive techniques.

Effect of semen thaw method on conception rate in four large commercial dairy heifer herds.

Semen processed with procedures intended to permit a flexible thaw method is used to breed millions of cows yearly. One method of thawing straws, the "pocket thaw" is used extensively with semen prepared with these procedures. Published field data is lacking for thaw method comparisons with semen processed to permit flexible-thawing. The objective of the present study was to measure the effect of semen thaw method (warm-water or pocket thaw) over all seasons and its interaction with herds, inseminators, straw package size, and sperm number on conception rate in commercial dairy heifer herds using semen processed with procedures historically optimized for success with flexible-thawing. Professional inseminators performed 11,215 services over a 16-month period in four large herds, achieving a 67.6% conception rate. Thaw method was alternated weekly. Thaw effect on conception status, determined by 70 days non-return rate, was estimated by a generalized linear mixed model. Neither thaw method nor number of sperm per straw significantly affected probability of conception (P=0.658 and 0.769, respectively). No interactions of thaw method with herd, sperm number, season, straw size, and straw size by season were detected (P=0.297, 0.526, 0.365, 0.723, and 0.824, respectively). Bull, herd, inseminator within herd, year, season, and straw size affected conception rate (P=0.002, 0.000, 0.000, 0.000, 0.000, and 0.014, respectively). In conclusion, for semen processed with procedures that permit flexible-thawing, thaw method (pocket thaw versus warm-water thaw) did not affect conception rate under commercial conditions and with routine semen handling methods.

Prospects for spermatogenesis in vitro.

In recent years, extraordinary progress has been made in a broad range of reproductive technologies, including spermatogonial transplantation in the male. However, effective procedures for the complete recapitulation of spermatogenesis in vitro, including meiosis, have remained elusive. Such procedures have the potential to facilitate (1) mechanistic studies of spermatogenesis, (2) directed genetic modification of the male germ line, and (3) treatment of male factor infertility. Early studies demonstrated the importance of germ cell-Sertoli association for germ cell survival in vitro. Recently, evidence for male germ cell survival and progression through meiosis has been reported for the rat, mouse, and man. We demonstrated the expression of spermatid-specific genes (protamine and transition protein 1) by alginate-encapsulate neonatal bull testis cells after 10 weeks in culture, suggesting that meiosis had occurred. Although identifiable germ cells in these cultures were very sparse, some indication of acrosome development was observed. Following round spermatid injection (ROSI) with presumptive spermatids produced in vitro, 50% of blastocysts produced were diploid and 37% were Y-chromosome positive. Improved culture conditions, which promote germ cell survival, differentiation, and proliferation, are essential for in vitro spermatogenesis (IVS) to become a useful technology. Other approaches to male germ cell manipulation and spermatid production are discussed.

In vitro production of haploid germ cells from fresh or frozen-thawed testicular cells of neonatal bulls.

Improved methods for culturing spermatogenic cells will facilitate the study of spermatogenesis, treatment of male factor infertility, and genetic modification of the male germ line. The objective of this study was to develop a procedure for achieving male germ cell progression through meiosis in vitro. Testes from 3-day-old bulls were decapsulated and seminiferous tubules were dissociated enzymatically to recover Sertoli and germ cells. Dissociated cells were reaggregated by phytohemagglutinin and encapsulated by calcium alginate, then cultured for up to 14 wk in modified Dulbecco modified Eagle medium/F12 (32 degrees C, 5% CO(2) in air). At 2, 5, and 10 wk, cultured cells were examined and evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis for protamine-2 (PRM-2) and transition protein-1 (TP-1) mRNA, expressed specifically in round spermatids. Ploidy was characterized by flow cytometric analysis of DNA content of cultured cells. Only Sertoli cells and gonocytes were observed in seminiferous tubules of 3-day-old testes. By 10 wk of culture, small spherical cells (7-10 microm) were apparent at the margin of cell associations in culture. Following RT-PCR and Northern blot analysis, specific bands corresponding to PRM-2 and TP-1 were detected only in adult testis RNA or after 10 wk of culture. Based on flow cytometry, a haploid population of cells appeared in vitro that was not in 3-day-old bull testis. The novel culture system developed in this study is the first to promote differentiation of gonocytes to presumptive spermatids in vitro based on the expression of spermatid-specific genes.

Sperm numbers inseminated in dairy cattle and nonreturn rates revisited.

Three experiments were conducted to test fertility when sperm numbers per insemination ranged from 10 x 10(6) to 40 x 10(6) total sperm. All semen was from Holstein bulls that were on a regular schedule of semen collection. The semen was extended with heated homogenized whole milk, cooled, glycerolated, and frozen according to standard procedures. Semen was distributed to a large group of inseminators to minimize differential field effects on treatment. All experiments were a randomized block design, including a split plot in Experiment 2. In Experiment 1, data for 31,399 first inseminations distributed among treatments of 20 x 10(6), 25 x 10(6), 30 x 10(6), and 40 x 10(6) total sperm resulted in 69.8, 70.0, 70.1, and 70.1% nonreturns at 59 d, respectively. In Experiment 2, data for 18,197 first inseminations divided over treatments of 12 x 10(6), 16 x 10(6), and 20 x 10(6) total sperm resulted in 70.2, 72.4, and 70.8% nonreturns at 59 d, respectively. In Experiment 3, 38,890 first inseminations distributed over treatments of 10 x 10(6), 13 x 10(6), 16 x 10(6), and 20 x 10(6) total sperm resulted in 70.5, 72.2, 73.1, and 71.5% nonreturns at 59 d, respectively. Bull nonreturns ranged from 64 to 76% in the three trials. These results indicate that, under good conditions, total sperm numbers per straw can be reduced to 10 x 10(6) total sperm with a reduction of nonreturn rates at 59 d, for most bulls, of about 1 percentage unit from the maximum when professional inseminators are use.

Categorical data analysis of the effect on bull fertility of butylated hydroxytoluene addition to semen extenders prior to freezing.

Butylated hydroxytoluene is an antioxidant that has antiviral properties and sustains sperm viability during freezing and thawing. A field trial involving 11 bulls and 19,000 AI was conducted to determine whether addition of .5 mM butylated hydroxytoluene to whole milk extender during seminal processing affected bull fertility as estimated by nonreturn rates generated by cows bred to the bulls. Effects of bull, batch of semen nested within bull, treatment, and month of AI were studied. Nonreturn rates were recorded for each month for every bull, batch of semen (ejaculates pooled on a given day), and treatment combination. Because some bulls had < 6 batches of semen, the original experimental design was reduced to two smaller designs. Categorical data analysis with maximum likelihood estimation was used for analysis of nonreturn rates. The results from three models were used to interpret the data. The nonreturn rates were approximately 73.9% for the butylated hydroxytoluene treatment and 74.1% for the control. In all models, bull effect was significant, but batch, month of AI, and treatment had no effect on bull fertility. Addition of .5 mM butylated hydroxytoluene to whole milk extender during semen processing did not affect bull nonreturn rates.

Semen quality and behavior of Holstein bulls exposed to estradiol-treated bulls for mounts.

The objectives were to test the effects of estradiol treatment of teaser bull mounts on sexual behavior and on quality and quantity of sperm obtained from sires managed as in large commercial AI breeding organizations. In a change-over design, the same teaser bulls were either untreated or treated with estradiol. Five semen-producing bulls were ejaculated twice per day on Tuesdays and Fridays after epididymal reserves were partially depleted. A 15-min period of continuous sexual preparation with three false mounts allowed was standard before each semen collection. All bulls were attracted to and licked the preputial area of the teaser bulls followed by the Flehmen response during the period of sexual preparation. Bulls usually completed the false mounts in < or = 15 min, and all thrusted vigorously with both hind feet moving forward synchronously at this time on 100% of the 80 semen collections. Major differences among bulls and between first and second ejaculates occurred in semen volume, semen concentration, and total sperm collected. An increase of 10% in total sperm output when bulls were exposed to treated teaser bulls could be of commercial benefit. The correlation between total time to first mount for the two ejaculates per bull each day and total sperm collected per bull per day was -.44. Thus, the shorter time to first mount may be useful as a low level predictor of higher sperm out-put per bull.

Fertility of bull spermatozoa frozen in whole milk extender with trehalose, taurine, or blood serum.

Fertility of bull semen processed in heated whole milk-glycerol control semen extender with various additives was compared in six field trials. The additive in field trials 1 and 2 was 25.6 g of trehalose/L of the glycerol fraction of whole milk. Whole milk was heated to 95 degrees C for 10 min, cooled, and filtered 1 d before use (trial 1) or 3 d before use (trial 2). In field trial 3, 3.0 g/L of taurine were added to the glycerol fraction of whole milk. In field trial 4, specially prepared bovine serum (15% vol/vol) was included in the glycerol fraction of whole milk. Field trials 5 and 6 were larger fertility studies with trehalose in extenders prepared the day before use (trial 5) and 1 and 3 d before use (trial 6). Control and treated semen were coded and distributed randomly over a large group of professional inseminators. The 59-d nonreturn rates for control and treated semen, respectively, were as follows: trial 1, 74.1 and 73.7%; trial 2, 71.3 and 73.1%; trial 3, 74.9 and 70.9%; and trial 4, 75.1 and 71.6%. No significant differences resulted in trials 1 to 3, but bovine serum decreased the non-return rate in trial 4. Trials 5 and 6 resulted in nonsignificant improvement in fertility with added trehalose. Thus, these additives, useful as cryopreservatives or membrane protectors in other systems, did not enhance the fertility of sperm frozen in whole milk.

Rapid determination of sperm cell concentration in bovine semen by flow cytometry.

A flow cytometric technique is described for determining sperm concentration in fresh or extended semen with improved accuracy, precision, repeatability, ease of conduct, and rapidity. The technique is designed to measure the ratio of a known number of fluorescent beads admixed with sperm stained with either acridine orange or propidium iodide. A significant advantage of the technique is the distinct resolution between sperm and other particles (e.g., somatic cells, fat droplets, and bacteria in the semen or extender) that interfere in other counting protocols. Field testing of this protocol over the past 3 yr has demonstrated its superiority over the Coulter counter, hemacytometer, and spectrophotometer for accuracy in counting sperm in extended semen and the accuracy of counting sperm in straws based on preextension spectrophotometric determination of sperm concentration. Sperm chromatin quality can be determined simultaneously with this sperm counting procedure. This approach to counting sperm provides an excellent procedure for quality control of sperm numbers in processed semen.

Effects of egg yolk-citrate and milk extenders on chromatin structure and viability of cryopreserved bull sperm.

Semen from four Holstein bulls was evaluated to compare effects of four extender treatments on postthaw semen quality. Extender fractions A and B, either heated whole milk or 20% egg yolk-citrate, were combined to yield the extender treatments 1) milk and milk, 2) milk and egg yolk-citrate, 3) egg yolk-citrate and milk, and 4) egg yolk-citrate and egg yolk-citrate. Semen was evaluated at thawing and after 30, 60, 120, and 180 min of incubation at 38.5 degrees C. Flow cytometry showed that acridine orange-stained sperm were most susceptible to in situ DNA denaturation when fraction A was milk. For sperm stained with rhodamine 123, flow cytometry showed that the proportion with intact mitochondrial membrane potential was lowest of all treatments at thawing but greatest at 180-min incubation with milk and milk extender. Flow cytometry of propidium iodine-stained sperm showed greatest proportion of cell membrane intact sperm when fraction A was egg yolk-citrate. Light microscopy showed the lowest proportion of cell membrane intact sperm with milk and milk extender after eosin-aniline blue vital staining. Postthaw motility scores tended to be reduced when both extender fractions were egg yolk-citrate. Results demonstrate differential extender effects on postthaw semen quality and indicate that altering extender composition or sequence of adding extender components may improve postthaw quality of cryopreserved sperm.

Comparison of semen quality in young and mature Holstein bulls measured by light microscopy and flow cytometry.

Random samples of cryopreserved, milk-extended semen, collected from 20 Holstein bulls at about 14 mo of age (young) and again at about 4 yr of age (mature), were evaluated at thawing and during 3-h incubation to compare semen quality of young versus mature bulls. Evaluation by differential interference contrast microscopy showed greater proportions of cytoplasmic droplets in semen from young versus mature bulls. Mature bulls exhibited greater proportions of intact acrosomes in freshly thawed semen than did young bulls. Evaluation of sperm chromatin structure by flow cytometry after staining with acridine orange showed lower values for mature versus young bulls, indicating resistance of DNA in nuclear chromatin to acid denaturation increased with age. Correlations between ages for most sperm morphology, acrosome integrity, and flow cytometry variables were high and positive. Nonreturn rate for young bulls was positively related to morphologically normal sperm and acrosomal integrity and negatively related to flow cytometry traits. Results suggest semen quality of young bulls was related to subsequent quality as mature bulls. With flow cytometry, differences were detected between semen samples that were not evident with light microscopy.

A new antibiotic combination for frozen bovine semen. 2. Evaluation of seminal quality.

Amikacin, clindamycin, gentamicin, Linco-Spectin, minocin and tylosin were added individually and in combinations at various concentrations to bovine neat semen and to egg-yolk citrate, egg yolk-tris or heated whole milk extenders (nonglycerol fractions) prior to final processing for freezing to -196 degrees C. After thawing samples, seminal quality was measured by progressive motility and acrosomal integrity evaluations. Studies were performed in parallel with microbiological efficacy studies of Shin et al. (7). The antibiotic combination including gentamicin, tylosin and Linco-Spectin at 500 ug/ml, 100 ug/ml and 300/600 ug/ml, respectively, was not detrimental to seminal quality and, in the parallel studies, was most efficacious in controlling microorganisms potentially present in bovine semen.

A new antibiotic combination for frozen bovine semen, 3. Evaluation of fertility.

Field fertility (nonreturn rate) studies were performed independently by three artificial insemination organizations to evaluate bovine semen processed for freezing using the antibiotics gentamicin, tylosin and Linco-Spectin at concentrations of 500 ug, 100 ug, and 300/600 ug, respectively, per milliliter of neat semen and per milliliter of nonglycerol portion of the extender. The antibiotic combination including penicillin, dihydrostreptomycin, polymyxin B sulfate, with/without Linco-Spectin (500 units/ml, 2000 ug/ml, 1000 units/ml and 300/600 ug/ml, respectively) was used as the control treatment. Results indicated no significant effect on seminal quality as measured by field fertility under the conditions of these experiments using heated whole milk or egg yolk-sodium citrate seminal extenders. Use of the new antibiotic combination has been adopted by Certified Semen Services.

Antibiotics for bull semen frozen in milk and egg yolk extenders.

In six experiments, gentamicin, clindamycin, amikacin, minocin, tylosin, and Linco-Spectin were tested for their effect on motility and fertility of frozen bull spermatozoa and all but clindamycin were used in fertility trials. Antibiotics were added to raw (unextended) semen and nonglycerol portions of the three commonly used semen extenders, whole milk, egg yolk-Tris, and egg yolk-citrate. Semen was frozen in .5-ml straws, stored in liquid nitrogen, and thawed at 37 degrees C for 30 s. Postthaw percentage of motile spermatozoa in antibiotic-treated semen was different for individual bulls in each experiment. Percentage of motile sperm was slightly but significantly depressed at the higher concentrations of clindamycin and Linco-Spectin tested in whole milk and with minocin in the two egg yolk extenders. In general, nonspermicidal concentrations of each antibiotic were established. Of five antibiotics tested for fertility, only gentamicin reduced fertility on the basis of 59-d nonreturn rates.

Whole milk extender with antibiotics to eliminate Campylobacter fetus from frozen bovine semen.

Fresh bovine semen was contaminated with five strains of Campylobacter fetus at 10(5) to 10(6) organisms per ml of semen. The contaminated semen was processed to contain 500 units of polymyxin B, 500 units of penicillin G, and 1000 micrograms of streptomycin sulfate per ml, incubated at 35 degrees C for 10 min, and added then to whole milk extender with like units of antibiotics for further processing. Both a low and high initial sperm count requiring extension rates 1:7 and 1:39 were studied. Specimens for bacterial viability were processed for culture prior to adding bacteria, 100 min after initial antibiotic treatment, prior to freezing, and after thawed semen was centrifuged and washed to remove extender and antibiotic residue. All strains of Campylobacter fetus were not viable 6 h postantibiotic treatment prior to freezing. All nonantibiotic treated controls were viable after thawing and washing. Removal of antibiotics from thawed semen by centrifugation and washing indicated that antibiotics had bactericidal rather than only bacteriostatic activities against Campylobacter fetus.

Unusual observations on a serologically negative bluetongue virus infected bull.

A Holstein bull named "Regency" born in New York in July 1968, maintained in an artificial insemination (AI) stud for 9 years, unremarkable from the point of view of health history or semen production, was discovered "by accident" to have bluetongue (BT) virus (BTV) in his semen. A microscopic study in 1975 and 1977 revealed unusual cytoplasmic vacuoles in the sperm of "Regency" prompting us to send semen to A. J. Luedke, USDA-ARS, Denver, Colorado, USA, to attempt virus isolation. Serotype 13 BTV was isolated from the semen. Serum sent with the semen was negative for antibody by the agar gel immunodiffusion (AGID) and complement fixation (CF) tests. Annual AGID serologic tests begun in 1975 and continuing to the present have been negative for antibody to BTV for all bulls in this AI stud. "Regency" was moved to Auburn, Alabama, USA in 1978 and further studies were performed. The bull was maintained with the Veterinary School herd. The BT serologic status of the cattle did not change during the year the bull was in the herd nor were the cows he bred or their calves serologically positive for BT. Studies on insemination of cattle with experimentally contaminated semen indicated that 5X10(4) infectious doses of BTV were required for infection. These results suggest that since "Regency's" semen did not infect cows, the semen probably contained less than this amount of virus. The rete testis was cannulated, rete fluid collected, a testis removed and virus isolation attempted.(ABSTRACT TRUNCATED AT 250 WORDS)