Supplementation of progesterone via controlled internal drug release inserts during ovulation synchronization protocols in lactating dairy cows.

Our objective was to determine the effect of exogenous progesterone (P4) during a timed artificial insemination (TAI) protocol on pregnancies per AI (P/AI) in dairy cows not previously detected in estrus. Lactating cows (n=3,248) from 7 commercial dairy herds were submitted to a presynchronization protocol (2 injections of PGF(2alpha) 14 d apart; Presynch), and cows in estrus after the second PGF(2alpha) received AI (EDAI; n=1,583). Cows not inseminated by 12 to 14 d after the second PGF(2alpha) injection were submitted to a TAI protocol (GnRH on d 0, PGF(2alpha) on d 7, and GnRH+TAI 72h after PGF(2alpha)). At onset of the TAI protocol, cows were balanced by parity and days in milk and assigned randomly to receive no exogenous P4 (control, n=803) or a controlled internal drug release (CIDR) insert containing 1.38g of P4 from d 0 to 7 (CIDR, n=862). Blood samples were collected at the second PGF(2alpha) injection of the Presynch and on the day of the first GnRH injection of the TAI protocol for P4 determination. When P4 in both samples was <1 ng/mL, cows were classified as anovular, whereas cows having at least 1 sample >or=1 ng/mL were classified as cyclic. Concentration of P4 at 11 to 14 d after AI was determined in a subgroup of cows (n=453) from 2 herds. Pregnancy was diagnosed at 40+/-5 and 65+/-5 d after AI. Proportion of cows inseminated on estrus after the second PGF(2alpha) injection of the Presynch protocol differed among herds (range=26.7 to 59.8%). Overall P/AI for EDAI cows at 40+/-5 and 65+/-5 d were 36.2 and 33.7%, respectively, and pregnancy loss was 8.8%. Proportion of cyclic cows at the onset of the TAI protocol differed among herds (range from 66.5 to 86.3%), but did not differ between treatments (control=72.4%, CIDR=74.1%). Treatment affected P/AI at 40+/-5 (control=33.3%, CIDR=38.1%) and 65+/-5 (control=30.0%, CIDR=35.1%) d after AI but did not affect pregnancy loss (8.6%). Cyclic cows had greater P/AI at 40+/-5 (38.2 vs. 29.3%) and 65+/-5 d (35.1 vs. 26.1%) after AI, but cyclic status had no effect on pregnancy loss. Treatment affected P4 concentration after AI, with more CIDR cows having P4 >or=1 ng/mL (94.4 vs. 86.9%) and P4 >or=3.2 ng/mL (81.8 vs. 68.0%) at 11 to 14 d after AI compared with control cows. Treatment of cows not previously detected in estrus with a CIDR insert during a TAI protocol increased proportion of cows with functional CL after AI and P/AI.

Superovulation and embryo recovery from peripubertal Holstein heifers.

The use of peripubertal donors in embryo transfer (ET) programs presents significant opportunity to accelerate genetic gain in domestic livestock by reducing the generation interval. These studies were designed to evaluate feasibility of superovulation and embryo recovery in peripubertal heifers (starting at 7.8 months of age), and to determine whether subsequent reproductive and lactational performance of donor heifers were impaired. Study 1 utilized 10 pairs of contemporary full-sibling heifers in which one heifer in each pair was assigned to receive a superovulation regimen and her full-sibling contemporary received placebo. Treated heifers were artificially inseminated at estrus and embryos were flushed transcervically 4-6 days later. Based on recovery of oocytes and/or embryos, 9 of 10 heifers responded to the hormonal regimen and 12 total embryos were recovered. Seven embryos (58%) were transferred into recipients resulting in five pregnancies. Control and treated heifers remained in the herd and were bred at a natural estrus by AI at 15 months of age. Lactation records, i.e., 305 days mature equivalent (305 d ME) were obtained, and all animals were evaluated for udder conformation traits between 32 and 38 months of age. Reproductive traits (age at first calving and days to conception) and lactational traits of heifers subjected to embryo transfer and their non-treated full-siblings did not differ (P > 0.05). Study 2 was conducted to establish the commercial feasibility of hormonally programming peripubertal heifers ranging in age from 7.8 to 9.9; 10 to 11.9; 12 to 13.9 and >/= 14 months. In total, 3982 embryos were recovered from 520 heifers, with 2419 (60.7%) of those categorized as viable (transferable). The number of ova/embryos obtained per flush (5.6 +/- 1.0) and the number of transferable embryos (2.8 +/- 0.5) was reduced (P < 0.05) in heifers of age 7.8-9.9 months compared to all other age groups. There was no difference (P > 0.05) in the number of ova/embryos recovered (7.8 +/- 0.3), or the number of transferable embryos (4.8 +/- 0.2), among heifers that were >/=10 months of age. The number of unfertilized ova did not differ by age, however, more degenerate embryos tended to be recovered from heifers <10 months of age compared to heifers >/=14 months of age. These data indicate that transferable embryos can be safely recovered from heifers beginning at 10 months of age without compromising subsequent reproductive or lactational performance of the donor.

Physiological responses in thermal stressed Jersey cows subjected to different management strategies.

The effects of cooling and recombinant bovine somatotropin (rbST) on milk yield, reproductive performance, and health of Jersey cattle during summer thermal stress were measured for 2 yr. Cows were assigned to one of two groups based upon days in milk (DIM), parity, and genetic index. Year 1 and year 2 control cows (n = 143, n = 183, respectively) were housed in a pen with only shades. Cooled treatment cows each year (n = 142, n = 180) were housed with a spray and fan system for evaporative cooling. Cows were assigned at various days postpartum, not before d 63, coincident with commencement of rbST injections. One half of cows in each group received rbST on d 63 postpartum. Cows were assigned to the shade trial ranging from d 63 to 190. Cooled versus noncooled DIM were similar at the start of the trial. Trials began on July 1, 1999, and July 1, 2000, and concluded on September 30, 1999, and September 25, 2000. The ANOVA of daily milk weight data was conducted utilizing a 2 x 2 factorial design with cooling and rbST treatments as main effects. Cooling in combination with rbST increased milk yield compared with no cooling and no rbST for 1999 and 2000 (25.5 versus 21.8 kg/d, and 23.7 versus 20.5 kg/d, respectively). In general, cooling improved health and reproductive performance.

Response of Holstein and Brown Swiss cows fed alfalfa hay-based diets to supplemental methionine at two stages of lactation.

In this study, we evaluated the effects of dietary supplementation at two stages of lactation with various levels of Mepron85 (M85) and M85 plus DL-methionine (DL-Met) on milk production and composition of Holstein and Brown Swiss cows fed an alfalfa-hay and corn grain-based diet. In experiment 1, control diets were formulated to supplement, in early lactation [days in milk (DIM) = 73.2], concentrations of metabolizable methionine at 104% of the estimated requirements based on the Cornell Net Carbohydrate and Protein System. Treatment groups were fed the control diet plus 10, 20, or 30 g/d of M85 at 116, 128, or 139% of the estimated requirements for metabolizable methionine. The supplementation with 10 g/d in Brown Swiss and 30 g/d of M85 in Holstein cows increased milk yields and fat percentage, but had no effects on protein percentage. These data suggested that the estimated postruminal supply of metabolizable methionine in the control ration was limiting for maximum milk fat synthesis. Conversely, in experiment 2, the cosupplementation with M85 (15 g/d) plus DL-Met (15 g/d) to cows in midlactation (DIM = 140.5) did not influence fat percentage, but increased protein yield and percentage (+0.1%) in both Holstein and Brown Swiss, and lactose percentage (+0.18%) in Holstein cows. The supplementation with 15 g/d of M85 reduced milk and protein yields, whereas 15 g/d of DL-Met reduced protein percentage in four of the five experimental weeks for Holstein cows. We conclude that supplementation with M85, alone or in combination with DL-Met, may be used to influence milk composition, but these effects are influenced by dosage and type of supplemental methionine, breed, and stage of lactation.

Identification of a heparin-binding protein in bovine seminal fluid as tissue inhibitor of metalloproteinases-2.

Presence or absence of three distinct bovine seminal heparin-binding proteins (21-31 kDa) recognized in sperm extracts by a monoclonal antibody, M1, is a diagnostic indicator of fertility differences among bulls producing normal semen. We recently identified a 31 kDa fertility-associated antigenin bovine seminal fluid as a unique DNase I-like protein. We now report purification and identification of a 24 kDa seminal heparin-binding protein (HBP-24) recognized by M1. N-terminal microsequence analysis of HBP-24 purified from seminal fluid yielded 20 amino acid residues that displayed 90% identity to the N-terminus of a bovine metalloproteinase inhibitor identified as tissue inhibitor of metalloproteinases-2 (TIMP-2). A single immunoreactive band migrating at 24 kDa was detected in Western blots of cauda epididymal sperm extracts following incubation with purified seminal heparin-binding proteins and subsequent washing in vitro, indicating TIMP-2 bound to sperm membranes. Expression of TIMP-2 mRNA was detected by RT-PCR in bovine bulbourethral gland, prostate, and seminal vesicles. Mobility of the 24 kDa heparin-binding protein increased under nonreducing SDS-PAGE to approximately 21 kDa, characteristic of the reported molecular mass of TIMP-2. To our knowledge, this is the first report of TIMP-2 binding to spermatozoa and of TIMP-2 mRNA expression in bovine accessory sex glands. These results corroborate previous reports regarding the site of production of heparin-binding proteins that are related to bull fertility, and suggest that TIMP-2 influences fertility of bulls, either through inhibition of metalloprotease activity in semen or via undefined activities independent of matrix metalloproteinase (MMP) inhibition.

Reproductive performance of Holstein cows receiving somatotropin.

Some dairy producers resist using bovine somatotropin (bST) beginning at 9 wk postpartum because of the concern that fertility is compromised. We conducted a trial with a total of 205 Holstein cows, 100 multiparous and 105 primiparous, to evaluate reproductive performance in two high producing herds in Arizona and southern California. Rolling herd averages for both herds for milk production exceeded 10,700 kg/yr. Data were collected for cows calving December 1996 through August 1997. The voluntary waiting period was 60 d postcalving, with cows randomly assigned to receive bST or no treatment (controls). In the 180-d interval after calving, 65.4% (68/104) of the control cows were diagnosed pregnant. With bST-treated cows, 48.5% (49/101) were pregnant in that same interval. A chi-square value from a linear model indicated that pregnancy outcome differed significantly between treatment groups. With a similar method of analysis, first-service conception rate was not significantly different between treatment groups. An extended voluntary wait and breeding interval is recommended for cows receiving bST, similar to suggestions from other published reports.

Artificial insemination outcomes in beef females using bovine sperm with a detectable fertility-associated antigen.

In this study, semen samples from 25 bulls that had passed a breeding soundness evaluation were analyzed for the presence or absence of a 31-kDa protein, known as fertility-associated antigen (FAA), on spermatozoal membranes. Eighteen bulls had FAA on sperm (FAA-positive) and seven were devoid of FAA on sperm (FAA-negative). A single ejaculate from each bull was extended and frozen with 25 to 30 x 10(6) sperm in .5-mL straws. Crossbred replacement heifers (n = 865) were estrus-synchronized and artificially inseminated either at timed AI or 12 h after they were detected in estrus. Mature cows (n = 285) were inseminated 12 h after they were detected in estrus during a 45-d AI period. Pregnancy rates (pooled) to first AI service for females (n = 764) inseminated with FAA-positive sperm were 65.6% and were 49.7% for females (n = 386) inseminated with FAA-negative sperm (P < .005). Among the estrus-synchronized replacement heifers, pregnancy rates to synchronized AI service for heifers (n = 550) inseminated with FAA-positive sperm were 62% and were 45.7% for heifers (n = 315) inseminated with FAA-negative sperm (P < .005). These data indicate that pregnancy rates to first AI service at spontaneous and synchronized estrus are higher when using semen from bulls with detectable FAA on spermatozoal membranes compared to semen from bulls devoid of FAA on membranes. Fertility-associated antigen is an important determinant for fertility potential of sperm from bulls to be used in AI breeding programs.

Purification and characterization of fertility-associated antigen (FAA) in bovine seminal fluid.

Heparin-binding proteins (HBP) recognized by a monoclonal antibody (M1) are produced by male accessory sex glands and bind to distinct regions of ejaculated bull sperm. Immunoblots of sperm proteins probed with M1 identified HBP variants of approximately 31-, 24-, and 21.5-kDa that were associated with increased fertility of bulls. The purpose of this study was to identify the 31-kDa HBP known as fertility-associated antigen (FAA). FAA was isolated by heparin-affinity chromatography and reversed-phase high performance liquid chromatography near homogeneity. Biochemical characterization indicated that FAA was an unglycosylated, basic protein. FAA protein was detected in seminal vesicle and prostate gland homogenates, and FAA extracted from sperm membranes by treatment with hypertonic media was identical biochemically to seminal fluid-derived FAA. N-terminal sequence analysis of purified FAA yielded a 26 amino acid sequence (L K I X S F N V R S F G E S K K A G F N A M R V I V) with 73% identity to a recently identified human deoxyribonuclease (DNase) I-like protein. Two internal amino acid sequences generated from lys-C digested FAA were 85% and 92% identical to the same DNase I-like protein. In conclusion, we have identified a bovine seminal heparin-binding protein that binds to sperm and is indicative of bull fertility as being similar to the family of DNase I-like proteins. These data demonstrate the presence of a novel DNase I-like protein in bull accessory sex glands and form the groundwork for the identification of a candidate genetic marker for fertility of bulls.

Pregnancy outcomes in two commercial dairy herds following hormonal scheduling programs.

There are a number of options for hormonal management of post partum dairy cows; however, only a few studies have made direct comparisons of these programs in commercial herd settings. We compared reproductive management programs of 2 commercial dairy herds to evaluate the efficacy of prostaglandin-based treatment regimens on reproductive outcomes. Cows in Herd A were left untreated and served as the negative controls. Cows in Herd B were given PGF2alpha every 14 d until first insemination beginning 45 d post partum and served as the positive controls. Treatment 1 (Ovsynch), initiated randomly during the estrous cycle, consisted of sequential injections of GnRH, PGF2alpha, GnRH again and insemination 16 to 20 h later. Treatment 2 consisted of an Ovsynch protocol, as described above, which was begun 7 d post estrus (Ovsynch + 7). In Herd A, the number of days from parturition to conception (days open) for controls, for Ovsynch and for Ovsynch + 7 were 126, 112 and 102, respectively. In Herd B, respective days open were 102, 100 and 93 for controls, Ovsynch and Ovsynch + 7. Hormonal intervention reduced the number of days open in both herds.

Fertility-associated antigen on bull sperm indicates fertility potential.

A 30-kDa heparin-binding protein named fertility-associated antigen (FAA) was identified in sperm membranes of beef bulls with greater fertility potential. In a survey of 2,191 beef bulls, 88% had FAA present in sperm membranes (FAA-positive), and 12% were FAA-negative. In the first study, 54 Santa Gertrudis and 51 Santa Cruz bulls were grouped (1 to 14 bulls per group) according to FAA profiles and were bred to 2,403 cows at ratios of 1 bull: 25 cows. Fertility for 14 groups of FAA-positive bulls averaged 88%, whereas three groups of FAA-negative bulls impregnated 79% of the cows. Thus, FAA-positive bulls were nine percentage points more (P < .01) fertile than FAA-negative bulls. In the second study, 2-yr-old Santa Cruz bulls (n = 26) were grouped according to FAA profiles and serving capacity. The fertility of the group of 12 high-serving-capacity, FAA-positive bulls was 87% of 270 cows. The group of six FAA-negative bulls with high serving capacity impregnated 78% of 143 cows. Among the groups of bulls with high serving capacity, FAA-positive bulls were nine percentage points more (P < .05) fertile than FAA-negative bulls. The group of eight FAA-positive bulls with low serving capacity impregnated the least (P < .01) percentage (69%) of 238 cows. Serving capacity of bulls should be considered when optimizing fertility potential. Among bulls with acceptable physical characteristics and serving capacity, determination of FAA profiles in sperm can be used as a tool to identify subfertile bulls.

Localization of a heparin-binding protein to distinct regions of bovine sperm.

Heparin-binding proteins (HBP) in bull seminal fluid bind to epididymal sperm membranes at ejaculation. Peptides recognized by a monoclonal antibody (M1) correspond to proteins identified in complexes that have the greatest affinity for heparin and are present on sperm from bulls with higher fertility. Presence of specific HBP on sperm regulates the ability of sperm to bind heparin, and heparin binding to sperm correlates with the fertility potential of a bull. In these studies, the interaction of HBP with sperm from 10 bulls of proven fertility was analyzed by immunofluorescence of M1 to determine the localization of heparin-binding proteins during capacitation, and the fluorescent binding patterns were compared to bull nonreturn rates. Immunofluorescent localization of M1 binding sites revealed the existence of specific membrane domains containing HBP in acrosomal and postacrosomal regions of ejaculated but not in epididymal sperm. Monoclonal antibody recognition of HBP localized on membranes of sperm revealed variable binding patterns of M1 to the acrosomal region in sperm from bulls of known fertility. Regression analysis indicated a negative relationship between sperm displaying exclusively acrosomal fluorescence and bull nonreturn rate. These data indicate that HBP bind to sperm in distinct patterns, one of which differed among bulls of varying fertility, and indicate no apparent relocalization of these sites during cellular changes that occur in preparation for fertilization.

Monoclonal antibody detection of heparin-binding proteins on sperm corresponds to increased fertility of bulls.

A monoclonal antibody (M1) was produced against seminal fluid heparin-binding proteins (HBP) from a vasectomized bull. In the first part of this study, the presence of HBP in sperm or seminal fluid was determined for 53 bulls with an ELISA using M1. Bulls (8 to 18 per pasture) were bred to 1,114 cows at ratios of 1 bull:25 cows. Bulls with detectable HBP on sperm membranes were 11 percentage points more fertile than bulls with undetectable HBP in sperm membranes. In the second part of this study, three sperm, membrane HBP approximately 30, 24, and 21.5 kDa were identified with Western blots using M1. Santa Gertrudis bulls (n = 64) were bred to 1,354 Santa Gertrudis cows in groups with 2 to 11 bulls. Bulls with those three HBP (Group A) or a single 30-kDa HBP (Group B) in sperm membranes had the greatest fertility, ranging from 74.4 to 89.9% (mean = 81.5%) of the palpated cows that were pregnant. Bulls with the 21.5- and 30-kDa HBP (i.e., the 24-kDa HBP was absent; Group C) had a reduced fertility of 61.3%. Bulls without detectable HBP (Group D) resulted in 41.9% of 186 cows palpated pregnant. Bulls in Groups A and B were more (P < .01) fertile than all other groups. In conclusion, the presence of HBP in sperm membranes was indicative of the fertility potential of bulls.

Fertility of range beef bulls grouped according to presence or absence of heparin-binding proteins in sperm membranes and seminal fluid.

Trials were performed to determine the relationship of heparin-binding proteins (HBP) to fertility of bulls. Red Angus (142), Santa Gertrudis (59), Gelbvieh (59), and Santa Gertrudis x Gelbvieh (40) bulls were identified according to the presence or absence of the greatest affinity HBP (HBP-B5) on sperm membranes and in seminal fluid. Nine to 20 bulls with the same HBP-B5 profiles were assigned to pastures with Santa Gertrudis cows at a ratio of 1 bull:25 cows. Fertility for Group 1 (80 bulls with HBP-B5 in sperm membranes but undetectable HBP-B5 in seminal fluid, in six pastures) was 82% pregnant of 1,692 cows. Group 2 bulls (48 bulls with HBP-B5 detectable in seminal fluid and in sperm membranes, in four pastures) impregnated 67% of 919 cows. Fertility for Group 3 (37 bulls with HBP-B5 in seminal fluid but undetectable HBP-B5 in the sperm membranes, in three pastures) and Group 4 (56 bulls with undetectable HBP-B5 in seminal fluid and sperm, in four pastures) was 63% pregnant of 747 and 1,208 cows, respectively. Group 1 had an average of 17% greater fertility compared with Groups 2, 3, and 4 (P < .05). In conclusion, groups with the greatest affinity HBP-B5 in sperm membranes but not in seminal fluid had greater fertility than did groups with other HBP-B5 profiles.

Modulation of bovine oocyte-cumulus cell complex maturation and fertilization in vitro by glycosaminoglycans.

Oocyte-cumulus cell complex maturation and fertilization potential of 4700 bovine oocytes in the presence of various intrafollicular regulators were studied. Endpoints for oocyte maturation included cumulus cell expansion, retention of [3H]hyaluronic acid, elevated cyclic AMP concentrations, and germinal vesicle breakdown. Media supplemented with FSH and fetal bovine serum stimulated oocyte maturation and fertilization. Heparin and mixed glycosaminoglycans (40% heparan sulfate and 60% dermatan sulfate) stimulated cumulus cell expansion and increased [3H]hyaluronic acid but decreased cyclic AMP and reduced percentages of oocytes with germinal vesicle breakdown at 12 h. Oocytes cultured in heparin or mixed glycosaminoglycans were incapable of fertilization. Addition of serum to oocytes treated with heparin decreased cumulus cell expansion. Addition of FSH to heparin and serum cultures did not overcome the inhibitory effect of serum on heparin-induced expansion. Dermatan sulfate failed to stimulate expansion of cumuli but resulted in cells retaining [3H]hyaluronic acid and elevated cyclic AMP content of oocytes. Dermatan sulfate did not affect the percentage of germinal vesicle breakdown at 12 h. Nitrogen-desulfated heparin did not promote any signs of oocyte maturation. These results demonstrate that heparin and mixed glycosaminoglycans stimulate events related to oocyte maturation but were not capable of preparing oocytes for fertilization. The addition of serum or desulfation of heparin inhibited heparin effects on oocyte maturation.

Validation and application of an assay for deoxyribonucleic acid to estimate concentrations of bull sperm.

Spectrophotometers are used for estimating sperm concentrations from raw ejaculates in semen processing laboratories. Unfortunately, these instruments have a limited detection spectrum and do not permit accurate quantification of sperm numbers in highly diluted or concentrated samples. The objectives of this study were to validate a DNA assay for quantification of sperm numbers in extended or undiluted semen samples and to determine precision of the assay. The principle of the assay is based upon a fluorescent dye that binds to adenine-thymine base pairs in double-stranded DNA. Semen samples and calf thymus DNA standards were sonicated in 2 M NaCl buffer with 1 mM EDTA. The DNA content of samples was compared to standards of calf thymus DNA using fluorometry. Sensitivity of the assay was determined to be 1.4 x 10(5) sperm cells. Concentrations of sperm estimated from DNA assay values did not differ from flow cytometric cell counts. Assays were performed in three different laboratories, using different equipment, to assess the assay's repeatability. Estimates of sperm concentrations determined by the DNA assay were similar, regardless of location and source of equipment used to perform the assays. This assay fulfills statistical criteria for being sensitive, accurate, and repeatable, and it can be employed in laboratories processing semen for artificial insemination as a tool for spectrophotometer calibration, a check for straw filling accuracy, or to quantify sperm numbers in extended, packaged semen.