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Contents The successful outcome of an insemination is a combination of both male and female fertility-linked factors. We investigated the first service conception rate of cows at artificial insemination ( AI) in the smallholder dairy farms in Bangladesh. Frozen straws were prepared from ejaculates of Bos indicus (n = 7) and Bos indicus × Bos taurus (n = 7) AI bulls. Fertility was determined from 6101 first services in cows that were performed by 18 technicians in four regions between April 2004 and March 2005. Pregnancy was diagnosed by rectal palpation between 60 and 90 days post-insemination. The Asian version of Artificial Insemination Database Application ( AIDA ASIA) was used for bulls-, cows- and AI-related data recording, and later retrieved for analysis. The mean ± SD number of inseminations performed from individual bulls and their conception rates were 436.0 ± 21.6 and 50.7 ± 1.9%, respectively. Logistic regression demonstrated body condition scores ( BCS), heat detection signs, months of AI and their interactions had greatest effects (odds ratios: 1.24-16.65, p < 0.04-0.001) on first service conception rate in cows. Fertility differed (p < 0.02-0.001) between the regions, previous calving months, months of AI, BCS, parity and heat detection signs of cows. Inseminations based on mounting activity (n = 2352), genital discharge (n = 3263) and restlessness and/or other signs (n = 486) yielded a conception rate of 53.6%, 48.8% and 50.1%, respectively (p < 0.05). Conception rate between technicians ranged between 43.4% and 58.6% (p < 0.05). The days interval from calving to first service (overall mean ± SD = 153.4 ± 80.6) had relationship (p < 0.001) with BCS, months of previous calving and parity of the cows. Fertility at AI in smallholder farms can be improved by training farmers on nutrition and reproductive management of the cows. [ABSTRACT FROM AUTHOR]

Fourier harmonic analysis (FHA) is a robust method for identification of minute changes in sperm nuclear shape that are indicative of reduced fertility. The current study was designed to develop a fertility prediction model for Nili-Ravi buffalo bulls through FHA of sperm. In experiment I, FHA technique was standardized, average sperm nuclear perimeter was measured and sperm nuclear shape plot of buffalo bull was constructed. Sperm of buffalo bulls (n = 10) were stained with YOYO-1 and Hoechst-33342 to differentiate live and dead, and digital images were captured using phase contrast and fluorescent microscopy. The images were analyzed by ImageJ software and 100 sperm/bull were evaluated. The results are described as mean ± SEM values of mean harmonic amplitude (mharm), skewness harmonic amplitude (skharm), kurtosis harmonic amplitude (kurharm) and variance harmonic amplitude (varharm) at Fourier frequencies 0-5 along with the cartesian and polar coordinate plots of buffalo bull sperm. In experiment II, a fertility prediction model was developed based on FHA of buffalo bull sperm. Semen samples of low (n = 6), medium (n = 3) and high (n = 8) fertility bulls were investigated for FHA of sperm and harmonic amplitudes (HA) were generated. Firstly, to determine if live and dead sperm population have unique nuclear shape distribution; the mean, skewness, kurtosis and variance HA 0-5 of 1700 live and 1294 dead spermatozoa of 17 bulls were evaluated. T-test signified a difference in the mharm0 (2.363 ± 0.01 vs. 2.439 ± 0.02), skharm0 (-0.0002 ± 0.07 vs. -0.266 ± 0.09), kurharm0 (-0.156 ± 0.07 vs. 0.260 ± 0.18), kurharm2 (0.142 ± 0.11 vs. 1.031 ± 0.32) and varharm4 (0.109 ± 0.00 vs. 0.082 ± 0.00) of live vs. dead sperm population (p < 0.05). Therefore, 100 live sperm/bull were further evaluated for mean, skewness, kurtosis and variance HA 0-5 values among high (n = 6) and low-fertility (n = 6) groups. Results of T-test showed higher values of mharm2 (0.739 ± 0.01 vs. 0.686 ± 0.00), mharm4 (0.105 ± 0.001 vs. 0.007 ± 0.001), and skharm0 (0.214 ± 0.109 vs. -0.244 ± 0.097) in high vs. low-fertility group (p < 0.05). In next step, five significantly different combinations of discriminant measures between high and low-fertility groups were obtained by discriminant analysis. In conclusion, mharm4, skharm0 and varharm2 correctly identified 91.7 % of bulls into their respective fertility groups, and upon cross validation the value of the canonical correlation was 0.928., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Most embryo losses occur in the first trimester of pregnancy in cows and include losses following embryo transfer. There is a resulting negative economic impact on cattle production systems when this occurs. Cellular and molecular mechanisms behind the maternal immune response to the growing embryo have not been fully characterized. The objective of this study was to examine the gene expression profiles of peripheral white blood cells (PWBCs) from pregnant cows 21 days after an embryo was transferred, and cows that were treated equally but lost the embryo. Specifically, we obtained and compared the transcriptome of PWBC from heifers that became pregnant at day 21 (N = 5) or failed to become pregnant after the embryo transfer (N = 5). Sequencing data can be accessed by Gene Expression Omnibus (GEO) with the accession number GSE210665. A total of 13,167 genes were evaluated for differential expression between groups. A total of 682 genes showed differential expression (p-value <.01), 302 genes were up-regulated while 380 were down-regulated due to pregnancy. The most significant genes were COL1A2, H2AC18, HTRA1, MMP14, CD5L, ADAMDEC1, MYO1A and RPL39, among others. Most of the significant genes are related to the up-regulation of inflammatory chemokine activity and immune defence response. Our findings extend the current knowledge that pregnancy alters the PWBC by promoting immune tolerance, cell chemotaxis, blood coagulation, angiogenesis, inflammatory response, cell adhesion and cytokine secretion. Our data suggest that pregnancy and ectoparasites could trigger poorly described genes in PWBC of cows, and a few previously escribed genes, such as IFI44. These results could shed light on the genes and mechanisms that promote tolerance to pregnancy and allow survival of the developing embryo., (© 2023 The Authors. Reproduction in Domestic Animals published by Wiley-VCH GmbH.)

Our objective was to evaluate reproductive management programs for submission of Holstein heifers for first insemination with conventional or sexed semen. In experiment 1, nulliparous Holstein heifers (n = 462) were submitted to a 5-d progesterone-releasing intravaginal device (PRID)-Synch protocol [d 0, GnRH + PRID; d 5, PGF 2α - PRID; d 6, PGF 2α ; d 8, GnRH + TAI] and were randomly assigned for PRID removal on d 5 or 6 of the protocol followed by timed artificial insemination (TAI) with conventional semen. Delaying PRID removal decreased early expression of estrus before scheduled TAI (0.9 vs. 12.2%), and pregnancies per AI (P/AI) did not differ between treatments. In experiment 2, nulliparous Holstein heifers (n = 736) from 3 commercial farms were randomized within farm to 1 of 3 treatments for first AI with sexed semen: (1) CIDR5 [d -6, GnRH + controlled internal drug release (CIDR); d -1, PGF 2α - CIDR; d 0, PGF 2α ; d 2, GnRH + TAI]; (2) CIDR6 (d -6, GnRH + CIDR; d -1, PGF 2α ; d 0, PGF 2α - CIDR; d 2, GnRH + TAI); and (3) EDAI (PGF 2α on d 0 followed by once-daily estrous detection and AI). Delaying CIDR removal decreased early expression of estrus before scheduled TAI (0.004 vs. 27.8%); however, CIDR5 heifers tended to have more P/AI at 35 (53 vs. 45 vs. 46%) and 64 (52 vs. 45 vs. 45%) days after AI than CIDR6 and EDAI heifers, respectively. Overall, CIDR5 and CIDR6 heifers had fewer days to first AI and pregnancy than EDAI heifers which resulted in less feed costs than EDAI heifers due to fewer days on feed until pregnancy. Despite greater hormonal treatment costs for CIDR5 heifers, costs per pregnancy were $16.66 less for CIDR5 than for EDAI heifers. In conclusion, delaying PRID removal by 24 h within a 5-d PRID-Synch protocol in experiment 1 suppressed early expression of estrus before TAI, and P/AI for heifers inseminated with conventional semen did not differ between treatments. By contrast, although delaying CIDR removal by 24 h within a 5-CIDR-Synch protocol in experiment 2 suppressed early expression of estrus before TAI, delaying CIDR removal by 24 h tended to decrease P/AI for heifers inseminated with sexed semen. Further, submission of heifers to a 5-d CIDR-Synch protocol for first AI tended to increase P/AI and decrease the cost per pregnancy compared with EDAI heifers., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Our objective was to determine the effect of increasing the interval from induction of ovulation to timed artificial insemination (TAI) on fertility by decreasing the interval from TAI to ovulation using sexed semen within a synchronized breeding program. Our hypothesis was that induction of ovulation earlier relative to TAI would increase pregnancies per artificial insemination (P/AI). Primiparous Holstein cows from 3 commercial dairy farms in the United States were submitted to a Double-Ovsynch protocol for first service as follows: Pre-Ovsynch (GnRH; 7 d, PGF 2α ; 3 d, GnRH), followed 7 d later by Breeding-Ovsynch [GnRH (G1); 7 d, PGF 2α ; 24 h, PGF 2α ], followed by the last GnRH treatment (G2), which varied between treatments, and TAI. To vary the interval between G2 and TAI, cows were randomized to 2 treatments to receive G2 either 16 (G2-16; n = 373) or 24 (G2-24; n = 357) h before TAI, which was fixed at 48 h after the second PGF 2α treatment of the Breeding-Ovsynch portion of the protocol. All cows were inseminated with sexed semen, and each herd used sires of their choosing, which were randomly allocated between treatments. Pregnancy diagnosis was conducted by herd veterinarians using transrectal ultrasonography. In disagreement with our hypothesis, G2-24 cows had fewer P/AI than G2-16 cows at 34 ± 3 d (44 vs. 50%) and 80 ± 17 d (41 vs. 48%) after TAI. Pregnancy loss (5 vs. 6%) and fetal sex ratio (92:8 vs. 90:10, female:male) did not differ between treatments for G2-16 and G2-24 cows, respectively. Thus, we reject our hypothesis and conclude that induction of ovulation earlier relative to TAI with sexed semen for first service after a Double-Ovsynch protocol decreased P/AI in primiparous Holstein cows., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Seasonal reproductive inefficiency is still observed in modern swine facilities. We previously reported global placental methylation activity was reduced from summer breedings and tended to be less from semen collected during cooler periods. The objective of the current study was to evaluate chromatin modification marks within swine placenta in relationship to breeding season, semen collection season, and semen storage. White composite gilts were artificially inseminated in August or January using single-sire semen that was collected during warm or cool periods and stored as either cryopreserved or cooled-extended. Gilts were harvested 45 days post-breeding, and placental samples from the smallest, average, and largest fetus in each litter were collected and stored at -80°C until RNA extraction. An RT 2 Profiler assay featuring 84 known chromatin modification enzyme targets was performed using placental RNA pooled by litter. Real-time quantitative polymerase chain reaction results were analyzed using the MIXED procedure, and P- values were Hochberg corrected using the MULTTEST procedure in SAS. The complete model included the fixed effects of breeding season (winter or summer), semen collection season (cool or warm), semen storage (cooled-extended or cryopreserved), interactions; boar as repeated effect; and plate as random effect. If interactions were not significant, only the main effects were tested. The genes, ATF2 , AURKA , and KDM5B , were different ( P < 0.05) by interaction of breeding season, semen collection season, and semen storage. In general, the greatest ( P < 0.05) expression was in placentas derived from summer breedings. Expression of AURKA was also influenced by semen collection and storage. Expression of placental KDM5B from winter breedings was also greater ( P < 0.05) from semen collected during cool periods. Placental expressions of ASH2L , DNMT3B , ESCO1 , HDAC2 , ING3 , KDM6B , MYSM1 , and SMYD3 were greater ( P < 0.05) from summer breedings. Increased expressions of known chromatin modification genes, from placentas derived from summer breedings, are likely responsible for differences in gene transcription between summer- or winter-derived placentas., (Copyright © 2020 Rempel, Parrish and Miles.)

Continuous progress in nanoscience has allowed the synthesis of various nanoscale particles, known as nanoparticles or nanomaterials which, by harnessing unique physico-chemical properties, are crucial for multiple bio-applications. Despite the revealed toxicity (nanotoxicity) of nanoparticles in various in vitro and in vivo studies, their careful design for biocompatibility and effective interactions with single-celled and multi-cellular organisms has permitted their use in several fields of research and biomedicine. The various nanoparticles synthesized and applied in the veterinary sciences, including reproductive biology, have shown potential to influence routine practices in animal production systems. These include post-collection manipulation of semen and the protection of high-quality spermatozoa to extend their preservation, and to improve sperm-related biotechnologies such as sperm-mediated gene transfer, sperm sorting, sex-sorting, and cryopreservation. Therefore, the application of nanotechnology-based tools to semen may enhance assisted reproductive technologies for biomedical applications and improve economic productivity for farmers. Here, we review the efficacy of available techniques and emerging tools of nanotechnology that might be useful for further selection of high quality boar spermatozoa and productivity improvement., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Epigenetics includes the study of external factors that can influence the expression of genes by altering the accessibility of DNA through methylation. To investigate the epigenetic influence of season, sperm head shape, and semen storage on placental and fetal tissues, pregnancies were generated in the summer or winter using boar semen from either least or most sperm head shape change, collected during cool or warm seasons, and stored as cooled-extended or cryopreserved. The lowest (p < 0.05) ratios of 5-methylcytosine to 5-hydroxymethylcytosine activity (5mC:5hmC) in fetal liver were from summer breedings and in placental tissues from winter breedings. The relative expression of placental CDH1 tended ( p < 0.10) to be greater in placenta generated from cryopreserved semen or semen collected during cool periods. The relative expression of placental GNAS was affected ( p < 0.05) by the interaction of breeding and semen collection seasons. Cryopreserved semen increased ( p < 0.05) the placental relative expression of GNAS. Placental MEST and RHOBTB3 tended ( p < 0.10) to have a greater relative expression from pregnancies generated using semen collected during cool periods used during winter breedings. Within fetal liver, the relative expression of GNAS and HGF was greater ( p < 0.05) from winter breedings. Interaction of winter breedings and least sperm head shape change tended ( p < 0.10) to have the greatest fetal liver expression of CDH1. Seasonality of semen collection, breeding, and the effect on sperm head shape change had an influence on the expression of genes with known differentially methylated regions or response to methylation activity from embryonic and extraembryonic tissues., (© 2019 Wiley Periodicals, Inc.)

The study of reproductive physiology in domestic ruminants has progressed from the whole animal to the molecular level in an amazingly short period of time. The volume of information on this subject is enormous; therefore, we have focused on domestic ruminants, with an emphasis on cattle. To date, artificial insemination (AI) is perhaps the most powerful technique that reproductive physiologists and geneticists have provided the livestock industry for genetic improvement. Early efforts to establish AI as a tool were initiated in Russia around 1899 and since that time major advances in methods of semen collection, evaluation of male fertility, cryopreservation of sperm, sex-sorted semen, and estrous cycle control have occurred. The preceding advances not only led to the widespread use of AI, but also contributed to our fundamental understanding of ovulation control, timing of insemination, gamete biology, and cryopreservation. In regards to anestrus, our understanding of the concept of neuroendocrine control of the pituitary gland and the role of steroid feedback led to the Gonadostat Theory, which proposes that onset of puberty is due to a decrease in the negative feedback of gonadal steroids over time. Subsequent studies in prepuberal and postpartum sheep and cattle established that a short luteal phase frequently precedes the first normal length cycle that is accompanied by estrous expression. This observation led to the common practice of treating prepuberal heifers and anestrous postpartum cows with a short-term progestin treatment (e.g., Controlled Internal Drug Release) to induce normal estrous cycles. In domestic ruminants, fertilization rate is high (85% to 95%); however, significant embryonic mortality before or around the time of maternal recognition of pregnancy (MRP) reduces the pregnancy rate to a single breeding. Significant effort has been directed at determining the time of MRP, the signal for MRP, as well as elucidating the physiological, cellular, and molecular dialogue between the conceptus and uterine environment. Advancements have now led us to the ability to edit the genome to alleviate disease and possibly improve production traits. In summary, major advancements in our understanding of reproductive biology have stemmed from efforts to establish the AI and embryo transfer technique and reduce the negative impact of anestrus and embryonic mortality in domestic ruminants.

Seasonal infertility is a limiting factor in boar fertility, and is increasingly important as climate changes. Spermatogenesis in the boar produces 256 spermatozoa per type A 1 spermatogonium, but the process is inefficient such that only 10-30% of these potential spermatozoa are actually produced. Heat further impacts spermatogenesis by reducing the number of specific germ cells produced while increasing the fraction of abnormal sperm. Early studies used whole-animal exposure to simulate seasonal exposure to heat under production settings, but this approach is associated with many confounding factors that make assessment of the mechanisms of heat-induced damage to spermatogenesis difficult. Scrotal insulation provides a better model to investigate the mechanisms and potential mitigation strategies of heat-induce damage. For example, scrotal insulation helped identify a link between short-term heat stress and damage to meiotic germ cells. This outcome is likely due to changes in the integrity of the blood-testis barrier, which induce apoptosis, autophagy and DNA damage in the germ cells. Further understanding how heat damages spermatogenesis, and whether or not this can be repaired, are crucial to mitigating heat effects on boars in production settings., (© 2017 Wiley Periodicals, Inc.)

As a result of research in the 1980s on in vitro maturation, sperm capacitation, and in vitro fertilization, the bovine is now one of the important models for development. Further, the current production of bovine embryos in vitro rivals that of in vivo embryo production for commercial applications. Researchers of today may be unaware of why decisions were made in the procedures. This review addresses the state of the art at the time of the work by Parrish et al. (Bovine in vitro fertilization with frozen thawed semen. Theriogenology 1986;25:591-600), and how later work would explain success or failure of competing procedures. Important was the use of frozen semen and heparin capacitation, because this allowed future researchers/practitioners to change sperm numbers and capacitation conditions to adjust for variations among bulls. The large numbers of citation of the original work stand the testament of time in the repeatability and success of the procedures. The work was done within the environment of the N.L. First laboratory and the unique interactions with a large number of talented graduate students, postdoctoral researchers, and technicians., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Consequences of heat stress exposure during the first 12 h of meiotic maturation differed depending on how and when bovine oocytes were activated. If heat-stressed oocytes underwent IVF at ~24 h, blastocyst development was less than for respective controls and similar to that obtained for nonheat-stressed oocytes undergoing IVF at 30 h (i.e. slightly aged). In contrast, if heat-stressed oocytes underwent chemical activation with ionomycin/6-dimethylaminopurine at 24 h, blastocyst development was not only higher than respective controls, but also equivalent to development obtained after activation of nonheat-stressed oocytes at 30 h. Developmental differences in chemically activated vs IVF-derived embryos were not related to fertilization failure or gross alterations in cytoskeletal components. Rather, ionomycin-induced calcium release and MAP kinase activity were less in heat-stressed oocytes. While underlying mechanisms are multifactorial, ability to obtain equivalent or higher development after parthenogenetic activation demonstrates that oocytes experiencing heat stress during the first 12 h of meiotic maturation have the necessary components to develop to the blastocyst stage, but fail to do so after fertilization.

The objectives of this study were to identify the stages of spermatogenesis susceptible to elevated testicular temperature in terms of sperm motility, viability, morphology, chromatin protamination and nuclear shape. The latter two valuable parameters are not included in routine semen analysis. Scrotal insulation (SI) was applied for 48 h in 2 Holstein-Friesian (HF) and 2 Belgian Blue (BB) bulls and semen was collected at 7 d intervals along with semen collection of a non-insulated bull of each breed. Semen samples were frozen and assigned to 4 groups: period 1 (preinsulation) = -7 d and 0 d, where 0 d = initiation of SI after semen collection; period 2 = 7 d (sperm presumed in the epididymis during SI); period 3 = 14 d to 42 d (cells presumed at spermiogenesis and meiosis stages during SI); period 4 = 49 d to 63 d (cells presumed at spermatocytogenesis stage during SI). The percentages of progressively motile and viable spermatozoa as assessed by computer-assisted sperm analysis (CASA) and fluorescence microscopy, respectively were decreased whereas abnormal sperm heads, nuclear vacuoles and tail defects were increased at period 3 (P < 0.05) compared to period 1, 2 or 4 in SI bulls of both HF and BB breeds. Protamine deficient spermatozoa as observed by chromomycin A(3) (CMA(3)) staining were more present (P < 0.05) at period 2 and 3 in both breeds compared to period 1 or 4. Sperm nuclear shape as determined by Fourier harmonic amplitude (FHA) was most affected by heat stress during period 3 (P < 0.01) and a higher response was observed in BB bulls than HF bulls. In conclusion, sperm cells at the spermiogenic and meiotic stages of development are more susceptible to heat stress. The lack of chromatin protamination is the most pertinent result of heat stress, together with subtle changes in sperm head shape, which can be detected by FHA but not by conventional semen analysis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Previously, we constructed an in vitro fertilization system for the identification of genes affecting fertility traits in dairy cattle. The efficiency of this system has been demonstrated by the identification of several genes affecting fertilization rate and early embryonic survival. However, to employ these genetic markers in marker- and gene-assisted selection programs, there is a need to validate in vitro results in phenotypic data sets collected in vivo. Thus, the objective of this study was to validate, in a population of Holstein bulls, the fertility trait genes we previously identified in an in vitro system. Estimated relative conception rate (ERCR) data from 222 Holstein bulls were obtained from 5 different artificial insemination companies in the United States. Bulls were genotyped for the genes FGF2, POU1F1, PRL, PRLR, GH, GHR, STAT5A, OPN, and UTMP, and the data were analyzed for association with ERCR using a mixed effects sire model. A stepwise model selection procedure revealed evidence of association with ERCR for FGF2 and STAT5A polymorphisms. The in vivo validation suggests that these genes can be used in gene-assisted selection programs for reproductive performance in dairy cattle. The genotypes found to be associated with low bull fertility in this study have been reported to be associated with high milk composition in previous studies. These findings provide molecular evidence for the antagonistic relationship between milk production and fertility observed for many years in different breeds of dairy cattle., (Copyright 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The functional relationships among intrafollicular free insulin-like growth factor 1 (IGF1), circulatory gonadotropins, and development of the dominant follicle were studied in 40 mares in two experiments. A GnRH antagonist (Acyline) was given i.m. at the expected beginning of follicular deviation (largest follicle or F1> or =20mm; Day 0) alone (Acyline group) or in combination with intrafollicular treatment of F1 with rhIGF1 (Acyline/IGF1 group). In Experiment 1, blood samples, follicular-fluid samples, and diameter of F1 were taken on Days 1 and 2. In Experiment 2, daily follicular diameter and blood samples were taken from Day 0 to ovulation. The GnRH antagonist induced a 50% decrease in circulatory FSH concentrations for 1 d and in LH for 2 d. In Experiment 1, control and Acyline/IGF1 groups had greater intrafollicular free IGF1 (P<0.05) and inhibin-A concentrations (P<0.08) than the Acyline group. The intrafollicular concentration of estradiol on Day 2 was greater (P<0.05) in the control group than in the Acyline and the Acyline/IGF1 groups. In Experiment 2, a decrease in diameter of F1 in the Acyline group was followed by a new follicular wave. All IGF-treated follicles grew and ovulated. Results indicated that the increase in intrafollicular free IGF1 observed in F1 in association with deviation is gonadotropin dependent. During the period of lesser gonadotropin concentrations from Acyline treatment, intrafollicular IGF1 stimulated follicular growth and inhibin concentrations, but not intrafollicular estradiol production., (Copyright 2009 Elsevier B.V. All rights reserved.)

The effects of FSH, LH or both on follicular growth and intrafollicular free insulin-like growth factor (IGF)-1 and oestradiol were investigated in mares after the beginning of deviation (largest follicle >/= 20 mm; Hour 0). A single treatment with a gonadotropin-releasing hormone antagonist (acyline) was given at Hour 3 to suppress the concentrations of FSH and LH. Five groups (n = 5 mares per group) were evaluated in the present study: (1) control; (2) acyline treated; (3) acyline + recombinant equine (re) FSH treated; (4) acyline + reLH treated; and (5) combined acyline + reFSH + reLH treated. Beginning at Hour 3, reFSH and reLH were given at 6-h intervals in eight decreasing or increasing doses, respectively. The reFSH and reLH prevented the acyline-induced decreases in FSH and LH, respectively. Diameters and concentrations of intrafollicular free IGF-1 and oestradiol of the two largest follicles at Hour 48 did not differ significantly between the control and acyline + FSH groups, but were reduced (P < 0.05) similarly in the acyline and acyline + LH groups. The combination of reFSH and reLH was no more effective than reFSH alone. The results demonstrate a role for FSH but not LH in the growth of the largest follicle and intrafollicular concentrations of free IGF-1 and oestradiol during the 48 h after the beginning of deviation in mares.

Background: Embryonic genome activation (EGA) is a critical event for the preimplantation embryo, which is manifested by changes in chromatin structure, transcriptional machinery, expression of embryonic genes, and degradation of maternal transcripts. The objectives of this study were to determine transcript abundance of HMGN3a and SMARCAL1 in mature bovine oocytes and early bovine embryos, to perform comparative functional genomics analysis of these genes across mammals., Results: New annotations of both HMGN3a and SMARCAL1 were submitted to the Bovine Genome Annotation Submission Database at BovineGenome.org. Careful analysis of the bovine SMARCAL1 consensus gene set for this protein (GLEAN&#95;20241) showed that the NCBI protein contains sequencing errors, and that the actual bovine protein has a high degree of homology to the human protein. Our results showed that there was a high degree of structural conservation of HMGN3a and SMARCAL1 in the mammalian species studied. HMGN3a transcripts were present at similar levels in bovine matured oocytes and 2-4-cell embryos but at higher levels in 8-16-cell embryos, morulae and blastocysts. On the other hand, transcript levels of SMARCAL1 decreased throughout preimplantation development., Conclusion: The high levels of structural conservation of these proteins highlight the importance of chromatin remodeling in the regulation of gene expression, particularly during early mammalian embryonic development. The greater similarities of human and bovine HMGN3a and SMARCAL1 proteins may suggest the cow as a valuable model to study chromatin remodeling at the onset of mammalian development. Understanding the roles of chromatin remodeling proteins during embryonic development emphasizes the importance of epigenetics and could shed light on the underlying mechanisms of early mammalian development.

Shortening age at puberty of crossbred breeding bull is an important issue in the tropics. This study aimed at selecting crossbred bulls at earliest possible age with bigger scrotum and potential for donating quality semen. One hundred and 31 pre-joining crossbred bulls of Central Artificial Insemination Laboratory, Saver, Dhaka were examined. The bulls being trained by seeing semen collection from mature bulls were allowed ejaculation into the artificial vagina at homosexual mount during a 20 min time at three occasions, every three months. Eighty one of 131 bulls produced at least one ejaculate during the study and their mean +/- SD age and scrotal circumference (SC) were 20.3 +/- 4.7 months and 28.2 +/-2.7 cm, respectively. Bulls' body weight, body condition score (BCS) and SC influenced the attainment of their puberty (p < 0.05). Bull's body weight had positive effects on scrotal circumference and ejaculate volume (p < 0.05). Scrotal circumference positively influenced the percentages of normal spermatozoa (p < 0.05). Scrotal skin-fold thickness negatively influenced the proportion of spermatozoa with normal head (p < 0.05). Based on age at first ejaculate and SC, 29.6% bulls (n = 24) were selected by cluster analysis. Selected bulls had mean +/- SD age 17.9 +/- 2.2 months, body weight 287.3 +/-48.6 kg, SC 30.5 +/- 1.5 cm, ejaculate volume 3.4 +/- 1.3 ml, sperm motility 50.8 +/- 17.2%, total spermatozoa per ejaculate 2541.9 +/- 1699.2 million and normal spermatozoa 81.5 +/-14.5%. The selected pubertal bull group was different from the unselected pubertal bulls at MANOVA (p < 0.0001). About 30% of pubertal crossbred bulls can be selected with shorter age and larger scrotum at puberty under conditions prevailed in Bangladesh.

Prostanoid synthesis via cyclooxygenase (COX)-2 induction during urothelial stretch is central to nociception, inflammation, contractility, and proliferation caused by urinary tract obstruction. We used our primary human urothelial cell stretch model published previously to evaluate the signaling mechanisms responsible for stretch-induced COX-2 expression in urothelial cells. To determine intracytosolic calcium concentrations ([Ca(2+)](i)), primary human urothelial cells were grown on flexible membranes and loaded with Fura-2 acetoxymethyl ester (AM). We determined [Ca(2+)](i) using a fluorescent scope during stretch. Additional cells were treated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, stretched, and COX-2 mRNA and protein were evaluated by real-time polymerase chain reaction and immunoblotting. To evaluate protein kinase C (PKC) in this system, cells were stretched and fractionated into membrane, cytosol, and nucleus. Fractions were immunoblotted for PKCalpha, beta1, and zeta, the predominant isoforms in urothelial cells. We treated additional cells with increasing concentrations of either bisindolylmaleimide-I or a peptide PKC pseudosubstrate inhibitor, and COX-2 mRNA and protein were evaluated after stretching. Furthermore, we transfected urothelial cells with siRNA against each of the inducible PKC isoforms in these cells and evaluated the stretch-induced COX-2 response. Stretch of urothelial cells activated calcium flux and PKC translocation to membrane and nucleus. Pharmacological inhibition indicated that stretch-induced COX-2 expression is dependent on calcium and PKC, and biochemical knockdown experiments indicated that PKCzeta is the predominant isoform mediating stretch-induced COX-2 expression. Elucidating the signaling mechanism of stretch-induced COX-2 expression may identify therapeutic targets.

Diverse groups of chemicals in culture media are needed for successful bovine oocyte maturation and embryo development during which dramatic cytoplasmic and nuclear reprogramming events take place. In vitro embryo production (IVP) procedures frequently include supplements such as serum and/or co-culture with various types of somatic cells. However, the presence of undefined serum in culture media introduces a variation from batch to batch, increases viral or prion contamination risk, and leads to problems during fetal development. The aim of the present study was to investigate the possibility of using chemically defined-synthetic serum substitute (SSS) in place of fetal calf serum (FCS) during maturation and long-term culture to stimulate in vitro maturation (IVM), fertilization (IVF) and subsequent embryo development. In Experiment I, the effect of the protein source on in vitro maturation was tested by maturing oocytes in culture media supplemented with 10% FCS (Control Group), 10% SSS (Group I) and 10% SSS+10 ng/ml epidermal growth factor (EGF) (Group II). In Experiment II, effects of SSS on both oocyte maturation and embryo development during in vitro culture (IVC) were tested by maturing oocytes in media supplemented with 10% FCS (FCS Group) or 10% SSS+10 ng/ml EGF (SSS Group), followed by IVF and IVC in SOF media supplemented with 10% FCS and 10% SSS on day 4 for FCS and SSS Groups, respectively. Even though rates for cleavage and development to blastocyst stage were not different, blastocyst cell numbers were higher in Group II containing SSS and EGF. The SSS supplementation group had higher apoptotic nuclei as compared to the FCS Group in Experiment II. Transcripts for heat shock protein 70 (Hsp70), interferon tau (IF-tau), DNA methyltransferase 3a (Dnmt3a), desmosomal glycoprotein desmocollin III (DcIII) and insulin-like growth factor II receptor (Igf-2r) were altered in different culture conditions in Experiment I. However, only glucose transporter-1 (Glut-1) mRNA was different in the SSS and FCS Groups in the second experiment. In summary, SSS and EGF in maturation medium and replacement of FCS with SSS alone in culture medium on day 4 of IVC support oocyte maturation and embryo development in vitro. However, significance of culture condition induced changes on the genome-wide abundance of messenger ribonucleic acid and the significance of the apoptotic nuclei during fetal development still remain to be determined.

Global activation of the embryonic genome is the most critical event in early mammalian development. After fertilization, a rich supply of maternal proteins and RNAs support development whereas a number of zygotic and embryonic genes are expressed in a stage-specific manner leading to embryonic genome activation (EGA). However, the identities of embryonic genes expressed and the mechanism(s) of EGA are poorly defined in the bovine. Using the Affymetrix bovine-specific DNA microarray as the biggest available array at present, we analyzed gene expression at two key stages of bovine development, matured oocytes (MII) and 8-cell-stage embryos, constituting the ultimate reservoir for life and a stage during which EGA takes place, respectively. Key genes in regulation of transcription, chromatin-structure cell adhesion, and signal transduction were up-regulated at the 8-cell stage as compared with 8-cell embryos treated with alpha-amanitin and MII. Genes controlling DNA methylation and metabolism were up-regulated in MII. These changes in gene expression, related to transcriptional machinery, chromatin structure, and the other cellular functions occurring during several cleavage stages, are expected to result in a unique chromatin structure capable of maintaining totipotency during embryogenesis and leading to differentiation during postimplantation development. Dramatic reprogramming of gene expression at the onset of development also has implications for cell plasticity in somatic cell nuclear transfer, genomic imprinting, and cancer.

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were cultured in vitro in three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR. In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P < 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P < 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P < 0.001). Expression of interferon tau (IF-tau) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P < 0.001). Gene expression did not differ between in vivo-derived blastocysts and their in vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

Cryopreservation causes a significant proportion of bovine oocytes to undergo degeneration during subsequent culture. We investigated the degeneration mechanism of cryopreserved oocytes. In vitro matured bovine oocytes were vitrified by the open-pulled straw (OPS) method. In each replicate, a group of oocytes were randomly taken after warming to determine oocyte survival by both morphological evaluation and propidium iodide vital staining. The remainders were evaluated by morphological criterion. Morphologically intact oocytes were co-incubated with frozen-thawed spermatozoa for subsequent development. In situ examination of DNA breaks in oocytes and embryos was conducted using a Fluorescein-FragEL DNA fragmentation detection kit. A caspase-3 detection kit was used to detect caspase-3 activity in oocytes and embryos. Most of the oocytes survived cooling and warming processes as assessed by both morphological evaluation and vital stain. During subsequent culture, some degenerating oocytes displayed observable apoptotic morphology, such as cytoplasmic condensation, cytoplasmic fragmentation, and formation of apoptotic bodies. Biochemical markers of apoptosis, such as apoptotic DNA fragmentation and activation of caspases, were detected not only in oocytes having typical apoptotic morphology, but also in oocytes without observable apoptotic morphology. In embryos, positive signals for both biochemical markers were detected in blastomeres. This experiment suggests that cryopreserved bovine oocytes degenerate via apoptosis during subsequent culture.

Developmental competence of mammalian oocytes is compromised by currently available oocyte cryopreservation protocols. Experiments were designed to examine the effect of three cryopreservation protocols on the integrity of bovine oocyte DNA. In vitro matured bovine oocytes were cryopreserved either by slow cooling, vitrification in 0.25 ml straws, or in open pulled straws. After thawing/warming, recovered oocytes were immediately subjected to morphological evaluation. Morphologically intact oocytes underwent comet assay to detect cryoinjury at DNA level. All cryopreservation protocols resulted in significant morphological damage as well as DNA damage compared to unfrozen control. Among the morphologically intact oocytes, there was no difference among protocols in the number of oocytes displaying DNA damage. However, oocytes that had been cryopreserved by slow cooling or by vitrification in open pulled straws exhibited more damage than those vitrified in 0.25 ml straws in the extent of DNA damage. If we combine the number of oocytes with morphological damage and oocytes with DNA damage, oocytes cooled by slow cooling resulted in the most damage. This experiment demonstrated that oocyte DNA is a target of cryoinjury and different protocols result in different degrees of damage., (Copyright 2003 Wiley-Liss, Inc.)

Two experiments in two seasons evaluated fertilization rate and embryonic development in dairy cattle. Experiment 1 (summer) compared lactating Holstein cows (n = 27; 97.3 +/- 4.1 d postpartum [dppl; 40.0 +/- 1.5 kg milk/d) to nulliparous heifers (n = 28; 11 to 17 mo old). Experiment 2 (winter) compared lactating cows (n = 27; 46.4 +/- 1.6 dpp; 45.9 +/- 1.4 kg milk/d) to dry cows (n = 26). Inseminations based on estrus included combined semen from four high-fertility bulls. Embryos and oocytes recovered 5 d after ovulation were evaluated for fertilization, embryo quality (1 = excellent to 5 = degenerate), nuclei/embryo, and accessory sperm. In experiment 1, 21 embryos and 17 unfertilized oocytes (UFO) were recovered from lactating cows versus 32 embryos and no UFO from heifers (55% vs. 100% fertilization). Embryos from lactating cows had inferior quality scores (3.8 +/- 0.4 vs. 2.2 +/- 0.3), fewer nuclei/embryo (19.3 +/- 3.7 vs. 36.8 +/- 3.0) but more accessory sperm (37.3 +/- 5.8 vs. 22.4 +/- 5.5/embryo) than embryos from heifers. Sperm were attached to 80% of UFO (17.8 +/- 12.1 sperm/UFO). In experiment 2, lactating cows yielded 36 embryos and 5 UFO versus 34 embryos and 4 UFO from dry cows (87.8 vs. 89.5% fertilization). Embryo quality from lactating cows was inferior to dry cows (3.1 +/- 0.3 vs. 2.2 +/- 0.3), but embryos had similar numbers of nuclei (27.2 +/- 2.7 vs. 30.6 +/- 2.1) and accessory sperm (42.0 +/- 9.4 vs. 36.5 +/- 6.3). From 53% of the flushings from lactating cows and 28% from dry cows, only nonviable embryos were collected. Thus, embryos of lactating dairy cows were detectably inferior to embryos from nonlactating females as early as 5 d after ovulation, with a surprisingly high percentage of nonviable embryos. In addition, fertilization rate was reduced only in summer, apparently due to an effect of heat stress on the oocyte.

An objective method for measuring bovine sperm nuclear shape was developed. Digital images of bovine sperm stained with propidium iodide were collected and Fourier functions used to describe the perimeters of individual sperm nuclei. Harmonic amplitudes from Fourier functions were first shown to be independent of sperm orientation during digitization. Sperm from 12 different bulls were used, and 6 harmonic amplitudes per sperm were found to adequately describe sperm nuclear shape. Based on harmonic amplitudes 0 to 5, cluster analysis was used to generate 20 different groups. Sperm within groups had similar morphologies and groups were distinguished by statistically unique shape characteristics. Harmonic amplitudes 0 to 5 can be used to distinguish previously reported abnormalities such as tapered, pyriform, macrocephalic, and microcephalic, as well as gradations in between. Furthermore, differences were detected among bull harmonic amplitude centroids (P < .05), indicating that bulls differ in mean sperm nuclear shape.

The relationship between sperm nuclear shape and bull fertility was determined. Two groups of bulls, 3 per group, were selected. Bulls differed in fertility based on lifetime nonreturn rates. Digital images of propidium iodide-stained sperm from each bull were collected and shape-evaluated by Fourier harmonic amplitudes 0 to 5. A discriminant function (P < .05) was constructed based on harmonic amplitudes and the 2 fertility groups. When individual sperm were classified as being of high or lower fertility, the percentage of each bull's sperm placed in the high-fertility group had a linear relationship (r = .89, P < .05) with fertility. To construct a plot of mean sperm shapes, a novel technique to automatically orient and identify the anterior tip of the sperm head was developed. The mean nuclear shape of high-fertility sperm was more elongated and tapered than those of lower fertility. A discriminant function (P < .05) was also constructed that separated the 6 bulls into 2 groups based only on the harmonic amplitudes or sperm nuclear shape. The bulls were correctly classified into the 2 fertility groups. A comparison of sperm chromatin structure analysis (SCSA) and harmonic amplitudes found that overall size variance, anterior roundness, and posterior taperedness of sperm nuclei were related to chromatin stability (P < .05). Some of the differences observed in sperm nuclear shape between the high- and lower-fertility bulls may be explained by varying levels of chromatin stability. However, sperm nuclear shape appears to contain additional information from chromatin stability alone. In this particular study, with 6 bulls, all with good chromatin quality, sperm nuclear shape was a better predictor of bull fertility.

A spectrophotometric assay was developed to measure the amount of esterase released from stallion spermatozoa. This assay was used to determine the percentages of capacitated stallion spermatozoa, determined by the ability of spermatozoa to undergo an acrosome reaction and release esterase in response to a lysophosphatidylcholine challenge, for spermatozoa incubated under conditions to increase intracellular calcium and cAMP. Incubation with 100 nmol calcium ionophore A23187 l(-1) induced 66% of stallion spermatozoa to capacitate after 60 min of incubation at 37 degrees C. Subsequent experiments investigating the effects of compounds that increase intracellular cAMP concentrations, 8-bromo cAMP (8bcAMP) and isobutyl-methylxanthine (IBMX), revealed that A23187 in combination with IBMX capacitated stallion spermatozoa after incubation for 240 min, while the combination of A23187 + 8bcAMP + IBMX capacitated spermatozoa in 40 min at 37 degrees C. Treating spermatozoa with a combination of compounds that increase intracellular calcium (A23187) and cAMP (8bcAMP and IBMX) capacitate stallion spermatozoa and may provide an efficient method to capacitate stallion spermatozoa for in vitro fertilization procedures.

To test the hypothesis that bull antisperm antibodies have the capacity to interfere with fertilization, antisperm antibodies were generated in three 13-mo-old Holstein bulls by auto-immunizing each bull with sperm three times. All bulls produced serum antisperm IgG1 and IgG2 antibodies. No serum antisperm IgA nor seminal plasma antisperm antibodies of any isotype could be detected by ELISA. Western blots were performed with immunopurified IgG1 and IgG2 from pre- and post-immunization sera from one test bull. Both post-immunization IgG1 and IgG2 recognized a 45-kDa sperm antigen. Serum samples from a normal bull stud population tested by ELISA had significantly higher levels of antisperm antibodies than did heifers. The bull stud serum samples giving the highest ELISA values differed from those of the immunized bulls in that their antisperm antibodies were of the IgM isotype only. Bull sperm were incubated with serum from the immunized and control bulls, then added to bovine oocytes in vitro. Incubation of sperm with post-immunization serum reduced in vitro fertilization rates (p < 0.01). This study demonstrated that antisperm IgG1 and IgG2 generated by sperm auto-immunizations reduced fertility in vitro, and therefore naturally occurring antisperm antibodies may affect fertility in bulls.

The development of successful methods of in vitro fertilization for bovine oocytes has advanced the bovine as a model for reproductive technology. The discovery of heparin as a capacitating agent has made it possible for investigators to have an inexpensive, readily available supply of bovine gametes for experimentation in reproductive biotechnologies such as gene transfer and cloning. The central event that mammalian sperm must undergo before being able to fertilize an oocyte is capacitation. Although we have methods which lead to efficient in vitro fertilization, we still lack understanding about the molecular mechanisms of capacitation. While numerous events occur during capacitation, it appears that regulation of intracellular Ca2+ (Ca(i)) is one of the most important. We found that the influx of Ca2+ into sperm during the first 2 hours of incubation is critical to heparin-induced capacitation. This is a period during capacitation when Ca(i) has not yet increased. We propose that during capacitation, the initial influx of Ca2+ into sperm is used to fill an intracellular Ca2+ store located in the acrosome. We found that thapsigargin, an inhibitor of an acrosomal Ca2+-ATPase, can stimulate capacitated sperm to acrosome react, trigger the opening of a store-operated calcium channel in the plasma membrane and has greater effects on capacitated sperm compared to noncapacitated sperm. An increase in intracellular Ca2+ was also detected in the anterior sperm head during capacitation, suggesting the loading of the acrosome with Ca2+. These observations may be important in the development of new methods for capacitation and understanding the death of sperm after cryopreservation.

Physiological levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17, 20-P) stimulated time- and dose-dependent increases in cortisol production by rainbow trout (Oncorhynchus mykiss) interrenal tissue cultured in vitro. Significant stimulation occurred in response to 100, 300, and 1000 ng/ml of 17,20-P. Lower doses were ineffective. Elevated cortisol levels were observed 1 hr after addition of 300 ng/ml 17,20-P. No additive or synergistic interaction was evident between human adrenocorticotropin fragment 1-24 (ACTH1-24) and 17, 20-P in stimulating cortisol secretion, although 300 ng/ml 17,20-P could further enhance cortisol production above levels already stimulated by 300 ng/ml ACTH. 17alpha, 20alpha-dihydroxy-4-pregnen-3-one also stimulated cortisol secretion, but was only half as effective as 17,20-P. Estradiol-17beta, testosterone, and 11-ketotestosterone had no effect on cortisol secretion. Inhibitors of mRNA and protein synthesis had no effect on 17,20-P-stimulated cortisol production. Radiotracer studies demonstrated that the bioconversion of 17,20-P to cortisol could fully account for the cortisol produced by the interrenal in response to 17,20-P and demonstrated that rainbow trout interrenal cells contain an active 20beta-hydroxysteroid dehydrogenase. These data suggest that 17,20-P may be a regulator of cortisol production during the periovulatory period in salmonid fishes.

Changes in the plasma membrane of bovine sperm during heparin-induced capcitation were detected by the binding of fluorescent labeled lectins to unfixed sperm. Of the seven lectins evaluated, only binding of wheat germ agglutinin (WGA) changed with capacitation. Sperm were classified into one of 5 patterns (p1-p5) based on staining with WGA, presence or absence of propidium iodide (PI) staining (dead or alive), and acrosomal integrity (acrosome intact or reacted). The major changes associated with capacitation occurred in p1 and p2. Sperm in p1 exhibited diffuse WGA binding over the anterior sperm head, were alive, and had intact acrosomes. In p2, sperm were also acrosome intact and alive, but lacked WGA binding. When sperm were incubated under capacitating conditions with heparin, there was a decrease over time in the percentage of sperm classified in p1 (p < 0.05) and an increase in the percentage of sperm in p2 (p < 0.05). When capacitation by heparin was delayed by the inclusion of glucose in the culture medium, the same heparin-dependent changes in the percentage of sperm in p1 and p2 were delayed (p < 0.05). When capacitation by heparin was inhibited by including protamine in the culture medium, the percentage of sperm in p1 or p2 was not different from sperm incubated without heparin. Heparin-induced capacitation was associated with a loss of WGA binding to the bovine sperm head.

Eight different lectins conjugated to fluorescein isothiocyanate (FITC) were used to screen for sperm plasma membrane changes during in vitro capacitation of bovine sperm. Analysis of lectin binding to sperm was done using flow cytometry. Of the eight lectins, only Triticum vulgaris (wheat germ agglutinin, WGA) binding to sperm was altered with capacitation. Capacitation of bovine sperm by heparin was found to decrease WGA binding to sperm by 78% (P < 0.05). The effect of capacitation by oviduct fluid was next compared with capacitation by heparin for changes in WGA binding to sperm. The effect of inhibiting capacitation with glucose on WGA binding was also determined. WGA-bound sperm were detected by flow cytometry as being present in two fluorescence peaks defined as low fluorescence (A) or high fluorescence (B) intensity. The percentage of sperm in peak A was greater for heparin and oviduct fluid-treated sperm compared to sperm incubated under noncapacitating conditions in only culture medium (P < 0.001). Capacitation with either heparin or oviduct fluid was inhibited by glucose as assessed by the ability of lysophosphatidylcholine (100 micrograms/ml) to induce acrosome reactions. Glucose also reduced the percentage of sperm in peak A for both heparin- and oviduct fluid-treated sperm (P < 0.01). We conclude that heparin or oviduct fluid induced changes on the sperm plasma membrane during capacitation. Binding sites for WGA on sperm were either structurally altered or lost during capacitation.

The objectives of these experiments were to characterize separation of frozen-thawed bovine spermatozoa on a Percoll gradient and then to compare sperm separation by either a swim-up or Percoll gradient procedure for the ability of spermatozoa to fertilize oocytes in vitro. The Percoll gradient was a 45 and 90% discontinuous gradient. Initial experiments found that centrifugation of semen on the Percoll gradient for 15 min at 700 g was sufficient to obtain optimal recovery of motile spermatozoa. Most of the nonmotile spermatozoa were recovered at the interface of the 45 and 90% Percoll layers, while the motile spermatozoa were primarily in the sperm pellet at the bottom of the gradient. When frozen-thawed semen from each of 7 bulls was separated by swimup, a mean +/- SEM of 9% +/- 1 of the motile spermatozoa were recovered after the procedure. In contrast, more spermatozoa were recovered after Percoll gradient separation (P < 0.05), with 40% +/- 4 of the motile spermatozoa recovered. The effect of separation procedure on in vitro fertilization found swim-up separated spermatozoa penetrated a mean +/- SEM of 74% +/- 5 of the oocytes, while fewer oocytes were penetrated by Percoll separated spermatozoa at 52% +/- 8 (P < 0.05). There was no effect of the separation procedure on the rates of polyspermy as measured by sperm/penetrated ova, with a mean +/- SEM of 1.25 +/-.09 for swim-up separated spermatozoa and 1.14 +/-.07 for Percoll separated spermatozoa (P>0.05). A carry over of Percoll into the fertilization medium with the Percoll separated spermatozoa was found not the cause for the decreased penetration of oocytes by these spermatozoa. In 2 of 3 bulls tested, the decreased penetration of oocytes by Percoll separated spermatozoa could be overcome by increasing the sperm concentration during fertilization from 1 x 10(6) to 5 x 10(6)/ml. When development of embryos fertilized by either swim-up or Percoll separated spermatozoa was compared for the semen from 2 bulls, a difference in cleavage rate was found in favor of swim-up separated spermatozoa (P < 0.05), but there was no effect of separation procedure on development (Day 7) to the morula + blastocyst or blastocyst stage (P>0.05). The disadvantages of the Percoll procedure could easily be overcome and the procedure was faster and yielded a six-fold greater recovery of motile spermatozoa than the swim-up method.

Equine oocytes (n = 537) were collected from slaughterhouse ovaries (n = 118 mares) by scraping the internal follicular wall. Preculture record was made of the appearance of oocyte investments (no cumulus, corona radiata only, compact cumulus, expanded cumulus), appearance of cytoplasm (homogeneous, condensed heterogeneous/fragmented), and nuclear maturation stages (germinal vesicle, germinal-vesicle breakdown, metaphase I, metaphase II, degenerated). There was no difference between follicles > 30 mm and follicles < or = 30 mm in the preculture frequency distribution among the 5 nuclear stages; 96% were at either the germinal vesicle or germinal-vesicle breakdown stages. Oocytes from follicles 5 to 30 mm were cultured in modified TCM-199 for 18, 24, 36 and 48 h. Postculture nuclear maturation classifications were immature (germinal vesicle, germinal-vesicle breakdown, and metaphase I), mature (metaphase II or secondary oocyte), and degenerated. The frequency distribution of oocytes among the 3 postculture maturation classifications changed (P < 0.05) at 18 h (15% mature oocytes), changed (P < 0.05) further at 24 h (55% mature oocytes), with no additional change for 36 or 48 h. The only preculture cytoplasm group that affected the postculture results was the heterogeneous/fragmentation group which had a high proportion of postculture degenerated oocytes (67%); however, only 4% of oocytes were in this group. Luteal status of the mare had an effect (P < 0.05) on the frequencies of the maturation classifications, but not enough to be useful in selecting oocytes. Consistency of the follicle and the type of oocyte investment did not alter significantly the maturation frequencies. The frequency of degenerated oocytes after culture was high under the following conditions: 1) diameter of the follicle from which the oocyte was selected was 5 to 10 mm (44% degenerated oocytes), 2) the largest follicle per pair of ovaries was < or = 10 mm (63%), and 3) the mare was pregnant (66%). These results were probably related to the reported high frequency of atretic follicles in the 5- to 10-mm population. In summary, oocytes from individual follicles < or = 10 mm or from follicles in which the largest follicle per mare was < or = 10 mm were the poorest candidates for in vitro maturation.

The effect of heparin-induced capacitation on the intracellular pH (pHi) of individual bovine sperm was determined with image analysis. Sperm were loaded with the acetoxymethyl ester of the pH sensitive fluorescent indicator, 2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF). The pHi of 5303 sperm was evaluated from a total of five bulls at .5, 2, 3, 4, and 5 h of incubation. The pHi did not differ between the sperm head and mid-piece (P > 0.05). An increase in sperm head pHi was seen in heparin-treated sperm at 3, 4, and 5 h of incubation relative to sperm incubated without heparin (control, P < 0.05). At 5 h of incubation, the pHi in heparin-treated sperm was 6.92 +/- 0.07, while control-treated sperm pHi was 6.70 +/- 0.03. Initially a normal frequency distribution was seen for sperm pHi in both heparin- and control-treated sperm. As the incubation progressed, the frequency distribution began to skew towards higher pHi in both samples but was more dispersed for the heparin-treated sperm. Following an NH4Cl-induced alkaline load, the pHi of both control- and heparin-treated sperm recovered toward the resting pHi with a half-time of recovery of 1.5-1.7 min. The recovery of sperm pHi was not due to leakage of NH4+ into sperm because recovery also occurred with trimethylamine. The instantaneous velocity of the pHi recovery (v(i)) was dependent on pHi and decreased as pHi decreased. Capacitation by heparin was associated with an 81% decrease in v(i) at a pHi of 7.00, but there was no effect of capacitation on the proton buffering power of the sperm, which was 87 +/- 8 mM/pH unit. Results demonstrate that both the regulation of pHi and resting pHi were altered during capacitation of bovine sperm by heparin.

The ontogeny of the interrenal stress response in rainbow trout was characterized by measuring resting and acute-stress-induced changes in whole-body cortisol levels in embryos and larvae at different early developmental stages. In Experiment 1, resting cortisol levels averaged 6.0 ng/g in newly fertilized eggs, fell to less than 0.3 ng/g by the time of hatching at Week 4 (incubation at 10 degrees), and increased to 1.4 ng/g by Week 5. Cortisol levels did not change in response to acute stress in 3-, 4-, or 5-week-old fish. In Experiment 2, resting cortisol averaged 1.4 ng/g in newly fertilized eggs, fell to less than 0.03 ng/g by Week 2, and then steadily increased between Weeks 3 and 6 to a peak of 4.8 ng/g before falling to 1.2 ng/g by Week 7. Cortisol levels did not change in response to acute stress in 3-, 4-, or 5-week-old fish. Six-week-old fish showed a 2.3-fold increase in cortisol levels at 1 hr poststress, indicating that the hypothalamic-pituitary-interrenal axis first develops responsiveness to stress 2 weeks after hatching and 1 week before the onset of exogenous feeding. The stress hyporesponsive period after hatching in rainbow trout may be homologous to the 2-week stress hyporesponsive period after birth in rodents, the function of which may be to maintain low, constant corticosteroid levels during a critical developmental period when these steroids can have permanent effects on neural organization. As suggested for mammals, this period may be a time when rainbow trout are particularly vulnerable to environmental effects on their subsequent development.

The objective of this study was to determine whether the initiation and length of the zygotic S-phase differs for embryos sired by bulls demonstrating high versus low fertility in vivo. Bovine oocytes were matured in vitro for 24-27 h and fertilized with frozen-thawed bovine semen. Six bulls that differed in fertility level were used in the study. The bulls were classified into two groups: those demonstrating high fertility in vivo (in vivo high-fertility bulls; n = 3), with a group mean +/- SEM lifetime nonreturn rate of 78 +/- 2%, and those demonstrating low fertility in vivo (low-fertility bulls; n = 3), with a group mean +/- SEM nonreturn rate of 69 +/- 1%. The S-phase in zygotes was identified by means of an immunocytochemical technique after pronuclear-stage zygotes were labeled with 5'bromo-2'deoxyuridine (BrdU). To visualize all pronuclei, presumptive zygotes were also stained with propidium iodide. In the first experiment, zygotes were labeled with BrdU at 2-h intervals from 8 to 20 h after sperm addition. There were no differences between bull fertility groups in the time course of pronuclear formation (p > 0.05). The beginning of S-phase was earlier in zygotes sired by high- compared to low-fertility bulls (p < 0.05). The end of S-phase was not affected by sire fertility group (p > 0.05). In the second experiment, zygotes were labeled with BrdU continuously from 8 to 20 h after sperm addition.(ABSTRACT TRUNCATED AT 250 WORDS)

Capacitation is an important maturational event in the life of a spermatozoan that allows the sperm to undergo a stimulus-induced acrosome reaction. Bovine sperm can be induced to undergo capacitation in vitro by heparin or oviduct fluid, and capacitation can be inhibited by glucose. We found that glucose did not interfere with 3H-heparin binding to sperm. Glucose inhibition of capacitation could be reversed in a dose-dependent manner by 8-bromo-cAMP or by the phosphodiesterase inhibitors isobutylmethylxanthine or caffeine, with ED50S of 25, 32, and 183 microM, respectively. The maximal effect of 8-bromo-cAMP on capacitation was during the first 2 h of a 4-h incubation. Sperm cAMP increased during capacitation with heparin from an initial value of 4.1 +/- 0.1 to 7.3 +/- 1.1 pmol cAMP/20 x 10(6) sperm at 4 h of incubation. Control sperm cAMP at 4 h increased only to 4.9 +/- 0.8 pmol cAMP/20 x 10(6) sperm. There were both similarities and differences in the characteristics of capacitation by heparin or oviduct fluid. Both glucose and protamine sulfate were found to suppress the heparin-dependent cAMP increase and inhibit capacitation. Capacitation by oviduct fluid was inhibited by either glucose or protamine sulfate. A small increase in sperm cAMP was associated with capacitation by oviduct fluid but was not affected by glucose or protamine sulfate.

The objective of this study was to investigate the importance of cAMP during capacitation of bovine sperm. The competitive antagonist of cAMP, Rp-adenosine-3'5'-cyclic monophosphorothioate (Rp-cAMP), blocked heparin-induced capacitation (p < 0.05). The effect of Rp-cAMP on heparin-induced capacitation was reversed by 8-bromo-cAMP. The maximal inhibitory effect on capacitation occuroff when Rp-cAMP was added at the start of sperm incubation. These results support an important role for cAMP-dependent protein kinases during heparin-induced capacitation of bovine sperm. Further support for a role for protein phosphorylation during capacitation came from the use of the protein phosphatase inhibitor, okadaic acid. Okadaic acid had no affect on heparin-induced capacitation of bovine sperm (p > 0.05); however, bovine sperm were capacitated by okadaic acid in the absence of heparin (p < 0.05). The relationship of cAMP to capacitation-associated changes in sperm intracellular pH (pHi) was investigated using image analysis of bovine sperm. The pHi of sperm increased during capacitation, and Rp-cAMP did not affect the change in pHi. We conclude that heparin-induced capacitation of bovine sperm involves an increase in cAMP and a protein phosphorylation event but that these do not induce the increase in pHi associated with capacitation.

A study was conducted to evaluate the potential of rescuing immature oocytes from the ovaries of an endangered wild bovid, the gaur (Bos gaurus). Recovered, immature gaur oocytes (n = 59) placed in culture were evaluated for: (1) nuclear maturation after 22 h of culture, (2) fertilization with either thawed homologous (gaur) or heterologous (Bos taurus) spermatozoa 18 h after insemination and (3) embryo development. Gaur oocytes (n = 6) evaluated by fixation and staining at 22 h had all matured to metaphase II in vitro. Insemination of gaur oocytes in vitro resulted in normal fertilization (defined as the presence of spermatozoa head or two pronuclei) and embryo development to the two- and four-cell stage of 53.6% (15 of 28) and 50.0% (9 of 18), respectively, using homologous spermatozoa. The incidence of normal fertilization of in vitro matured (IVM) gaur oocytes with heterologous spermatozoa was 53.8% (7 of 13). Insemination of domestic cow oocytes in vitro resulted in normal fertilization and embryo development of 41.7% (45 of 108) and 60.0% (12 of 20), respectively, using heterologous spermatozoa. Two of four gaur embryos (50%) developed to the blastocyst stage by day 7. Embryo transfer of these two conspecific gaur blastocysts into two Holstein recipients resulted in one confirmed pregnancy. One live-born calf was delivered by Caesarean section 308 days after embryo transfer. These results demonstrate the potential of combined IVM and IVF for recovering immature germplasm from an endangered species. Specifically, immature gaur ovarian oocytes are capable of in vitro maturation and fertilization with thawed homologous spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic and biochemical approaches have contributed to an explosion of literature on cell-cycle control. Regulation of the cell-cycle is controlled by a series of kinases and phosphatases. Key control points are during the G(1)-S and G(2)-M transitions. During both transitions, cyclins interact with a specific kinase to allow a cell to pass through that phase. The meiotic maturation of oocytes, fertilization and embryo development are all events influenced by cell-cycle regulation. Understanding cell-cycle control should provide new ways for gamete and embryo biologists to approach culture and development problems.

The proportion of transferable beef embryos obtained after superovulation, follicle aspiration, and in vitro maturation and fertilization has been small. To seek possible explanations, cows on different planes of nutrition were treated with exogenous gonadotropin and oocytes were isolated from their ovaries. The record for each oocyte included characteristics of the follicle, ovary, and cow from which it was obtained and the response to in vitro maturation, fertilization, and development. The sample was used to obtain estimates of the relationships among the variables. The logistic function with the probability of normal development as the dependent variable was the basic equation of the statistical model. When an explanatory variable was itself a result of the biological system, an equation explaining variation therein was added to the model. Had equations representing endogenous regressors not been added to the model a simple, single equation would have represented oocyte development response; given an oocyte at aspiration only one variable, cumulus quantity, was found to condition the probability of normal development directly. However, the complete model included four additional equations: 1) the probability that an oocyte was recovered at aspiration was conditional on the plane of nutritional treatment and progesterone concentration in follicular fluid; 2) cumulus quantity was conditional on the presence on a corpus luteum, follicle size, and progesterone concentration; 3) progesterone concentration was dependent on plane of nutrition; and 4) corpus luteum was conditional on plane of nutrition. The estimated model provided some insight into the complexity of oocyte development response and the role nutrition may play.

Bovine cumulus-oocyte complexes (COC) (n = 4230) were used in this study to assess the effects of culture method, hormonal supplementation, and cumulus cell concentration on maturation, fertilization and development of resulting embryos. Five treatments were evaluated. 1) 10 COC/50-microliter drops under oil in TCM 199 supplemented with 10% heat-treated fetal calf serum, follicle-stimulating hormone (0.5 microgram/ml), luteinizing hormone (5 micrograms/ml), and estradiol-17 beta (1 microgram/ml); 2) as in 1 without hormones; 3) as in 1 but in 3 ml TCM-199 in petri dishes without paraffin oil; 4) as in 2 but only 1 COC/50-microliter drop; and 5) as in 1 but with denuded oocytes. After 24 h maturation, the frequencies of oocytes reaching metaphase II were 98, 84, 92, 93, and 87%, respectively, for the five treatments. In the same order, percentages of normal fertilization were 73, 70, 62, 81, and 62%, and the frequencies of embryos containing two or more blastomeres at 65 h postinsemination were 69, 82, 66, 51, and 43%. The same five treatments were used in a second study in which 3,199 oocytes were fertilized, allowed to cleave in vitro to the 2- to 3-cell stage (42 h postinsemination), and transferred to oviducts of sheep (one treatment/oviduct) for 4 days. The frequencies of morulae or blastocytes obtained were 28, 18, 23, 24, and 11% for the five treatments, respectively. After nonsurgical transfer to bovine recipients (n = 8) using fresh or frozen-thawed embryos, three pregnancies past 50 days were obtained. Only one went to term with the birth of a live heifer calf.

The number of bovine spermatozoa separated in a swim-up procedure was quantified using an electronic cell counter. In an initial test of the swim-up procedure, non-frozen sperm samples with different ratios of live to dead cells were prepared and tested for the number of spermatozoa counted by the swim-up procedure. In ejaculates from six bulls, the number of spermatozoa swimming up was related to the number of live cells present (R2 = 0.97). Next, sperm quality of frozen-thawed semen immediately after thawing was measured at 37 C by swim-up sperm count, sperm motility, spermatozoa with an intact acrosome and migration in polyacrylamide gel and then compared with the fertility of the semen used for artificial insemination. Twenty-nine ejaculates of frozen-thawed semen from 11 bulls were evaluated. Correlations with fertility were highest on an ejaculate basis for motility (r = 0.41, P = 0.05) and for swim-up sperm count (r = 0.35, P = 0.06). On a bull basis, swim-up sperm count had the highest correlation with fertility (r = 0.59, P = 0.06). In a multiple regression model to predict male fertility that included all described measures of semen quality, a R2 value of 0.69 was obtained. This is the first report showing that the ability of spermatozoa to swim out of a more dense medium (whole milk-glycerol extender) into culture media is quantitatively related to in vivo fertility.

Bovine sperm incubated with heparin for 7.5-8.5 h underwent an acrosome reaction in the absence but not the presence of glucose (5 mM). When sperm were incubated under capacitating conditions with heparin for 4 h, glucose inhibited sperm penetration of oocytes (p less than 0.01) and lysophosphatidylcholine (LC) induced acrosome reactions. Addition of glucose for the last 0.25 h of a 4.25-h incubation with heparin had no effect on ability of sperm to acrosome-react in response to LC. Nonmetabolizable sugars 3-O-methyl glucose, 2-deoxyglucose, sucrose, and sorbitol did not inhibit capacitation as judged by sperm sensitivity to LC or fertilization (p greater than 0.05), but capacitation was inhibited by the glycolyzable substrates glucose, mannose, and fructose (p less than 0.05). The glycolytic inhibitor, fluoride, reversed glucose inhibition of capacitation in a dose-dependent manner similar to its effect on glucose uptake by sperm. Extracellular pH declined from 7.4 to 7.2 during a 4-h incubation of sperm with heparin and glucose. The decline of extracellular pH during sperm incubation with glucose did not affect capacitation, since only an extracellular pH below 7.02 inhibited capacitation. The intracellular pH (pHi) of sperm increased 0.40 units over a 5-h incubation under capacitating conditions. The change in pHi was inhibited by glucose. Incubation of sperm with heparin and glucose for 12 h resulted in capacitated sperm as judged by both LC sensitivity and fertilizing ability. These studies demonstrate that glycolyzable substrates delay capacitation of bovine sperm and suggest the effect is in delaying an alkalinization of pHi.