Effects of an anti-gonadoliberin releasing hormone vaccine on testicular, epididymal and spermatogenic development in the horse.

The effects of the GnRH vaccine Improvac® on testicular and epididymal morphometrics, histology and spermatogenesis were measured in 19 young (15-20 months) colts randomly assigned to one control (saline, castration at 57 days, n = 6) or either of two GnRH vaccine-treatment groups, T-57 (castration at 57 days, n = 7) or T-100 (castration at 100 days, n = 6), respectively. All were immunized on Day 0 with a single booster on Day 28. Excised testes and epididymides were weighed and processed for histology to measure tubule, epithelial and muscle dimensions, the ratio of interstitial tissue to seminiferous tubules and determine the stage of spermatogenesis. Testis volume, unchanged within controls, decreased in T-57 and T-100 groups by 50% and 70%, respectively. Treated colts' testes were significantly lighter than controls (64% relative difference); however, epididymal mass showed no significant differences between groups. Proportionally less seminiferous tubule relative to interstitial tissue was observed in both treatment groups (5%) versus controls (22%) with a mean tubule size 28% smaller than controls. Controls exhibited a high proportion of seminiferous tubules with advanced stages of spermatogenesis, whereas treated colts showed a high proportion of tubules in the early stages of spermatogenesis. In conclusion, immunization against GnRH in prepubertal colts was effective at reducing the development of their intra-scrotal reproductive organs and preventing normal spermatogenesis. GnRH vaccination of young colts effectively and consistently reduced testis mass, tubule size and relative proportion of seminiferous tubule tissue while retarding spermatogenesis. The epididymis showed changes with a smaller tubule diameter, lower epithelial height and thicker muscle layer recorded in treated compared to control colts.

Mitochondrial DNA replication is initiated at blastocyst formation in equine embryos.

Intracytoplasmic sperm injection is the technique of choice for equine IVF and, in a research setting, 18-36% of injected oocytes develop to blastocysts. However, blastocyst development in clinical programs is lower, presumably due to a combination of variable oocyte quality (e.g. from old mares), suboptimal culture conditions and marginal fertility of some stallions. Furthermore, mitochondrial constitution appears to be critical to developmental competence, and both maternal aging and invitro embryo production (IVEP) negatively affect mitochondrial number and function in murine and bovine embryos. The present study examined the onset of mitochondrial (mt) DNA replication in equine embryos and investigated whether IVEP affects the timing of this important event, or the expression of genes required for mtDNA replication (i.e. mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and single-stranded DNA binding protein (SSB)). We also investigated whether developmental arrest was associated with low mtDNA copy number. mtDNA copy number increased (P<0.01) between the early and expanded blastocyst stages both invivo and invitro, whereas the mtDNA:total DNA ratio was higher in invitro-produced embryos (P=0.041). Mitochondrial replication was preceded by an increase in TFAM but, unexpectedly, not mtPOLB or SSB expression. There was no association between embryonic arrest and lower mtDNA copy numbers.

Strong and stable geographic differentiation of swamp buffalo maternal and paternal lineages indicates domestication in the China/Indochina border region.

The swamp type of the Asian water buffalo is assumed to have been domesticated by about 4000 years BP, following the introduction of rice cultivation. Previous localizations of the domestication site were based on mitochondrial DNA (mtDNA) variation within China, accounting only for the maternal lineage. We carried out a comprehensive sampling of China, Taiwan, Vietnam, Laos, Thailand, Nepal and Bangladesh and sequenced the mtDNA Cytochrome b gene and control region and the Y-chromosomal ZFY, SRY and DBY sequences. Swamp buffalo has a higher diversity of both maternal and paternal lineages than river buffalo, with also a remarkable contrast between a weak phylogeographic structure of river buffalo and a strong geographic differentiation of swamp buffalo. The highest diversity of the swamp buffalo maternal lineages was found in south China and north Indochina on both banks of the Mekong River, while the highest diversity in paternal lineages was in the China/Indochina border region. We propose that domestication in this region was later followed by introgressive capture of wild cows west of the Mekong. Migration to the north followed the Yangtze valley as well as a more eastern route, but also involved translocations of both cows and bulls over large distances with a minor influence of river buffaloes in recent decades. Bayesian analyses of various migration models also supported domestication in the China/Indochina border region. Coalescence analysis yielded consistent estimates for the expansion of the major swamp buffalo haplogroups with a credibility interval of 900 to 3900 years BP. The spatial differentiation of mtDNA and Y-chromosomal haplotype distributions indicates a lack of gene flow between established populations that is unprecedented in livestock.

Maternal age and in vitro culture affect mitochondrial number and function in equine oocytes and embryos.

Advanced maternal age and in vitro embryo production (IVP) predispose to pregnancy loss in horses. We investigated whether mare age and IVP were associated with alterations in mitochondrial (mt) DNA copy number or function that could compromise oocyte and embryo development. Effects of mare age (<12 vs ≥12 years) on mtDNA copy number, ATP content and expression of genes involved in mitochondrial replication (mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and mitochondrial single-stranded DNA-binding protein (SSB)), energy production (ATP synthase-coupling factor 6, mitochondrial-like (ATP-synth_F6)) and oxygen free radical scavenging (glutathione peroxidase 3 (GPX3)) were investigated in oocytes before and after in vitro maturation (IVM), and in early embryos. Expression of TFAM, mtPOLB and ATP-synth-F6 declined after IVM (P<0.05). However, maternal age did not affect oocyte ATP content or expression of genes involved in mitochondrial replication or function. Day 7 embryos from mares ≥12 years had fewer mtDNA copies (P=0.01) and lower mtDNA:total DNA ratios (P<0.01) than embryos from younger mares, indicating an effect not simply due to lower cell number. Day 8 IVP embryos had similar mtDNA copy numbers to Day 7 in vivo embryos, but higher mtPOLB (P=0.013) and a tendency to reduced GPX3 expression (P=0.09). The lower mtDNA number in embryos from older mares may compromise development, but could be an effect rather than cause of developmental retardation. The general down-regulation of genes involved in mitochondrial replication and function after IVM may compromise resulting embryos.

Dose-response effects of estrogenic mycotoxins (zearalenone, alpha- and beta-zearalenol) on motility, hyperactivation and the acrosome reaction of stallion sperm.

BACKGROUND: The aim of this study was to investigate the in vitro effects of the Fusarium fungus-derived mycotoxin, zearalenone and its derivatives alpha-zearalenol and beta-zearalenol on motility parameters and the acrosome reaction of stallion sperm. Since the toxic effects of zearalenone and its derivatives are thought to result from their structural similarity to 17beta-estradiol, 17beta-estradiol was used as a positive control for 'estrogen-like' effects. METHODS: Stallion spermatozoa were exposed in vitro to zearalenone, alpha-zearalenol, beta-zearalenol or 17beta-estradiol at concentrations ranging from 1 pM - 0.1 mM. After 2 hours exposure, motility parameters were evaluated by computer-assisted analysis, and acrosome integrity was examined by flow cytometry after staining with fluoroscein-conjugated peanut agglutinin. RESULTS: Mycotoxins affected sperm parameters only at the highest concentration tested (0.1 mM) after 2 hours exposure. In this respect, all of the compounds reduced the average path velocity, but only alpha-zearalenol reduced percentages of motile and progressively motile sperm. Induction of motility patterns consistent with hyperactivation was stimulated according to the following rank of potency: alpha-zearalenol > 17beta-estradiol > zearalenone = beta-zearalenol. The hyperactivity-associated changes observed included reductions in straight-line velocity and linearity of movement, and an increase in the amplitude of lateral head displacement, while curvilinear velocity was unchanged. In addition, whereas alpha- and beta- zearalenol increased the percentages of live acrosome-reacted sperm, zearalenone and 17beta-estradiol had no apparent effect on acrosome status. In short, alpha-zearalenol inhibited normal sperm motility, but stimulated hyperactive motility in the remaining motile cells and simultaneously induced the acrosome reaction. Beta-zearalenol induced the acrosome reaction without altering motility. Conversely, zearalenone and 17beta-estradiol did not induce the acrosome reaction but induced hyperactive motility albeit to a different extent. CONCLUSIONS: Apparently, the mycotoxin zearalenone has 17beta-estradiol-like estrogenic activity that enables it to induce hyperactivated motility of equine sperm cells, whereas the zearalenol derivatives induce premature completion of the acrosome reaction and thereby adversely affect stallion sperm physiology. The alpha form of zearalenol still possessed the estrogenic ability to induce hyperactivated motility, whereas its beta stereo-isomere had lost this property.

Clathrin is essential for meiotic spindle function in oocytes.

In the mammalian ovary, oocytes are arrested at prophase of meiosis I until a hormonal stimulus triggers resumption of meiosis. During the subsequent meiotic maturation process, which includes completion of the first meiotic division and formation of the second metaphase spindle, oocytes acquire competence for fertilization. Recently, it was shown that clathrin, a cytosolic protein complex originally defined for its role in intracellular membrane traffic, is also involved in the stabilization of kinetochore fibers in mitotic spindles of dividing somatic cells. However, whether clathrin has a similar function in meiotic spindles in oocytes has not been investigated previously. Our results show that endogenous clathrin associates with the meiotic spindles in oocytes. To study the function of clathrin during meiotic maturation, we microinjected green fluorescent protein-tagged C-terminal and N-terminal dominant-negative clathrin protein constructs into isolated porcine oocytes prior to in vitro maturation. Both protein constructs associated with meiotic spindles similar to endogenous clathrin, but induced misalignment and clumping of chromosomes, occurrence of cytoplasmic chromatin and failure of polar body extrusion. These data demonstrate that clathrin plays a crucial role in meiotic spindle function in maturing oocytes, possibly through spindle stabilization.

Expression of leptin receptor mRNA in cumulus cells is correlated with expression of PTX3.

This study investigated the role of the leptin system in human oocyte maturation and its prognostic value for IVF outcome. The protein concentrations of leptin and soluble leptin receptor in follicular fluid were determined and the free leptin index (FLI) were established. Additionally, mRNA expression levels of different leptin receptor (ObR) isoforms and of PTX3 and HAS2 in cumulus cells were quantified, mutually compared and analysed relative to FLI, body mass index, age and number of retrieved oocytes. Expression of all target genes was detected in the cumulus cells, with relatively low concentrations of ObR-Long. Strong mutual correlations were found between mRNA expression levels of leptin receptor isoforms (P < 0.001) and also between the short isoforms of the leptin receptor and PTX3 (P < 0.001). Although the mean values of the pregnant and non-pregnant groups did not differ significantly for any of the variables, the chance that treatment resulted in ongoing pregnancy was higher with leptin 0.5 ng/mg protein compared with concentrations >0.5 ng/mg protein (P < 0.05). It is concluded that the leptin system appears to play a role in the IVF protocol, whereby signal transduction in cumulus cells occurs predominantly via the short isoforms of ObR.

Functionality of cryopreserved juvenile ovaries from mutant mice in different genetic background strains after allotransplantation.

The rapid expansion of mutant mouse colonies for biomedical research has resulted in lack of space at laboratory animal facilities and increasing risks of losing precious lines. These challenges require cheap and effective methods in addition to freezing embryos and sperm to archive the expanding mutant mouse lines. Cryopreservation of mouse ovarian tissue has been reported, but the application in the diverse mutant lines and genetic backgrounds has not yet been studied. In this study, juvenile ovaries (10-day-old) collected from genetically modified mouse lines were cryopreserved using high concentrations of cryoprotectants (dimethyl sulfoxide (Me(2)SO) and ethylene glycol (EG)) and instrumented ultra-rapid freezing. The validation of the frozen ovary batches was assessed by orthotopically transplanting a thawed ovary into a nearly completely ovariectomized mature female (congenic with the ovary donor). After 2 weeks of recovery, the ovary recipient was continuously paired with a male (congenic with the ovary donor) to evaluate the fertility of the recipient and delivered offspring were genotyped to evaluate the continued functionality of the grafted ovary. The recipient females delivered genetically modified offspring starting 6 weeks after ovary transplantation and lasting up to 6 months. The presented cryopreservation and transplantation protocols enabled retrieval of the genetic modification in 20 (from 22) genetically modified mutant mouse models on a C57BL/6 (17), FVB (2), or BALB/c (1) background. The thawed ovaries functioned after successful orthotopic allotransplantation to congenic wild-type recipients and produced mutant offspring, which allowed recreation of the desired genotype as a heterozygote on the proper genetic background. The results indicate that cryopreservation of mouse ovaries is a promising method to preserve genetic modification of the increasing number of mutant mouse models and can be used as a model for ovary cryopreservation using a variety of mouse mutants.

CDC2/SPDY transiently associates with endoplasmic reticulum exit sites during oocyte maturation.

BACKGROUND: Mammalian oocytes acquire competence to be fertilized during meiotic maturation. The protein kinase CDC2 plays a pivotal role in several key maturation events, in part through controlled changes in CDC2 localization. Although CDC2 is involved in initiation of maturation, a detailed analysis of CDC2 localization at the onset of maturation is lacking. In this study, the subcellular distribution of CDC2 and its regulatory proteins cyclin B and SPDY in combination with several organelle markers at the onset of pig oocyte maturation has been investigated. RESULTS: Our results demonstrate that CDC2 transiently associates with a single domain, identified as a cluster of endoplasmic reticulum (ER) exit sites (ERES) by the presence of SEC23, in the cortex of maturing porcine oocytes prior to germinal vesicle break down. Inhibition of meiosis resumption by forskolin treatment prevented translocation of CDC2 to this ERES cluster. Phosphorylated GM130 (P-GM130), which is a marker for fragmented Golgi, localized to ERES in almost all immature oocytes and was not affected by forskolin treatment. After removal of forskolin from the culture media, the transient translocation of CDC2 to ERES was accompanied by a transient dispersion of P-GM130 into the ER suggesting a role for CDC2 in redistributing Golgi components that have collapsed into ERES further into the ER during meiosis. Finally, we show that SPDY, rather than cyclin B, colocalizes with CDC2 at ERES, suggesting a role for the CDC2/SPDY complex in regulating the secretory pathway during oocyte maturation. CONCLUSION: Our data demonstrate the presence of a novel structure in the cortex of porcine oocytes that comprises ERES and transiently accumulates CDC2 prior to germinal vesicle breakdown. In addition, we show that SPDY, but not cyclin B, localizes to this ERES cluster together with CDC2.

The effects of growth factors on in vitro-cultured porcine testicular cells.

Cell lines from neonate porcine testis were cultured and characterized and the effect of growth factors were investigated, in order to determine the requirements for the establishment of porcine male germ cell lines. In primary cultures, three different colony types with distinctive morphologies could be recognized. From colonies resembling mouse spermatogonial stem cells (SSCs), two cell lines were derived and maintained for nine passages after which proliferation stopped. Growth of these cell lines depended on the growth factors leukemia inhibitory factor (LIF), epidermal growth factor (EGF), glial derived neurotrophic factor (GDNF), and fibroblast growth factor (FGF). In both cell lines NANOG, promyelocytic leukemia zinc-finger (PLZF), and EPCAM, were expressed at higher levels and GFRA1, ITGA6, and THY1 at lower levels than in neonate porcine testis. Primary cultures of neonate pig testis were subjected to a factorial design of the growth factors LIF, GDNF, EGF, and FGF. EGF and FGF had a positive effect on the number and size of the SSC-like colonies. Addition of EGF and FGF to primary cell cultures of neonate pig testis affected the expression of NANOG, PLZF, POU5F1, and GATA4, whereas effects of LIF or GDNF could not be detected. FGF decreased the expression levels of NANOG, a marker for pluripotency also expressed in neonatal porcine male germ cells. FGF decreased expression of PLZF and enhanced the expression of pluripotency-related gene POU5F1 and Sertoli cell marker GATA4. EGF had a positive effect on PLZF expression levels and counteracted the positive effect of FGF on GATA4 expression. These results suggest that FGF can impede successful derivation of porcine SSCs from neonate pig testis.

Induction of the ovulatory LH surge in Asian elephants (Elephas maximus): a novel aid in captive breeding management of an endangered species.

A unique feature of the reproductive physiology of Asian elephants (Elephas maximus) is the occurrence of two LH surges before ovulation, instead of one. An anovulatory LH (anLH) surge, the function of which is unknown, occurs consistently 3 weeks before the ovulatory LH (ovLH) surge that induces ovulation. Thus, the ability to induce an ovLH surge would be useful for scheduling natural mating or artificial insemination. The present study tested the efficacy of a gonadotrophin-releasing hormone agonist (GnRH-Ag) to induce LH surges during the follicular phase of the oestrous cycle, which resulted in varied LH responses, but generally none were as high as previously documented natural surges. Thus, for the ovulation-induction trials, nine females were administered 80 microg GnRH-Ag intravenously at three time periods during the oestrous cycle, namely the anovulatory follicular phase, the ovulatory follicular phase and the luteal phase. During the late anovulatory follicular phase, nine of 10 females (90%) responded with an immediate LH surge followed 15-22 days later by an ovLH surge or a post-ovulatory increase in progestagens. In contrast, despite responding to the GnRH-Ag with an immediate increase in LH, none of the females treated during other periods of the oestrous cycle exhibited subsequent ovLH surges. One cow got pregnant from natural mating following the induced ovLH surge. In conclusion, ovLH induction is possible using a GnRH-Ag, but only during a specific time of the anovulatory follicular phase.

Enhancement of Bovine oocyte maturation by leptin is accompanied by an upregulation in mRNA expression of leptin receptor isoforms in cumulus cells.

In this study, the mechanisms of supposed leptin action on oocyte maturation were examined. Expression of leptin mRNA, as determined with RT-PCR, was present in oocytes but not in cumulus cells. The long isoform of the leptin receptor (ObR-L) was expressed exclusively in cumulus cells after 7 and 23 hr of maturation. In oocytes the expression of the short receptor isoform (ObR-S) and all the receptor isoforms combined (ObR-T) did not change during maturation, as determined by quantitative RT-PCR, but in cumulus cells there was a significant increase in ObR-S transcripts after 7 hr of maturation. To determine if leptin plays a role in resumption of meiosis, oocytes meiotically arrested by the connection of the cumulus to a piece of granulosa layer were exposed to leptin. After 23 hr of culture, the proportion of oocytes that had resumed meiosis did not differ from the control. Exposure of COCs to leptin (1,000 ng/ml) resulted after 17 hr of maturation in a smaller proportion of oocytes that was still in metaphase-I stage (M-I) compared to the control group. Fertilization of oocytes after maturation in the presence of leptin resulted in a larger proportion of embryos that had developed to the 8-cell stage on Day 5 compared to the control group and in more blastocysts on Day 8 of culture. It is concluded that leptin enhances meiotic maturation of bovine oocytes, and that this effect is cumulus cell-mediated.

Expression and localization of the mu-opioid receptor (MOR) in the equine cumulus-oocyte complex and its involvement in the seasonal regulation of oocyte meiotic competence.

The micro-opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT-PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cytoplasm of oocytes and cumulus cells. In vitro oocyte maturation rate (MR), analyzed by confocal microscopy for nuclear chromatin, microfilaments and microtubules, was reduced after the addition of 3 x 10(-8) M beta-endorphin in medium without additional hormones. Inhibitory effects of 10(-3) M Naloxone in oocytes collected in anestrous and spring transition were observed, both in presence and absence of hormones added to culture medium. Increased MRs were observed in oocytes collected in anestrous and cultured in presence of 10(-8) M Naloxone. The exposure to 10(-3) M Naloxone induced significant intracellular calcium increases in cumulus cells recovered all over the year. beta-Endorphin 3 x 10(-8) M induced significant calcium increases only in cumulus cells recovered in fall transition and anestrous. Naloxone 10(-8) M did not induce intracellular calcium modifications. We conclude that the MOR is differentially expressed in equine cumulus-oocyte complexes in the different seasons of the year and plays a role in the seasonal regulation of meiotic competence of equine oocytes.

Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos.

BACKGROUND: In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction. RESULTS: In order to identify stably expressed genes for normalisation of quantitative data, within early stages of development, transcription levels were examined of 7 frequently used reference genes (B2M, BACT, GAPDH, H2A, PGK1, SI8, and UBC) at different stages of early porcine embryonic development (germinal vesicle, metaphase-2, 2-cell, 4-cell, early blastocyst, expanded blastocyst). Analysis of transcription profiling by geNorm software revealed that GAPDH, PGK1, S18, and UBC showed high stability in early porcine embryonic development, while transcription levels of B2M, BACT, and H2A were highly regulated. CONCLUSION: Good reference genes that reflect total RNA content were identified in early embryonic development from oocyte to blastocyst. A selection of either GAPDH or PGK1, together with ribosomal protein S18 (S18), and UBC is proposed as reference genes, but the use of B2M, BACT, or H2A is discouraged.

Exposure of bovine sperm to pro-oxidants impairs the developmental competence of the embryo after the first cleavage.

The present study describes the effects of exposure of bovine sperm to mild and more intense ROS generating conditions. The membrane integrity of the incubated sperm was assessed and the incubated sperm were used for IVF after which the percentages of cleavage and blastocyst formation were determined for a period up to 9 days. The incubated sperm samples showed increased levels of molecular oxidation in the plasma membrane, the mitochondria, the cytosol and to a lesser extent in the sperm's DNA. The sperm membrane integrity as well as the first cleavage rates obtained with sperm from mild ROS generating conditions (100 microM H2O2) were not different from sperm incubated without pro-oxidants. However, exposure of sperm to more severe oxidative stress (500 mM H2O2 or a combination of 100 microM ascorbic acid, 20 microM FeSO4 and 500 microM H2O2) led to plasma membrane oxidation, reduced percentages of cleaved embryos and a reduction in the percentages of cleaved embryos that developed to the blastocyst stage. From these results, we conclude that the impact of oxidative stress to sperm becomes primarily manifest after the first cleavage of the formed zygote. Importantly, the level of lipid peroxidation in the sperm plasma membrane significantly correlates with blastocyst formation when the corresponding sperm is used for in vitro fertilization of oocytes.

Developmental competence of bovine oocytes after specific inhibition of MPF kinase activity: effect of estradiol supplementation and follicle size.

In the bovine, the concentration of 17beta-estradiol (E2) in the follicular fluid of the dominant follicle is high, indicating a possible role of E2 on the cytoplasmic maturation that occurs before the LH surge. The aim of this study was to investigate the role of E2 on the developmental competence of bovine oocytes originating from different sized follicles and temporarily maintained at the germinal vesicle stage with roscovitine (ROS). First, the efficiency of ROS to inhibit germinal vesicle breakdown (GVBD) in oocytes harvested from small (3-4 mm diameter) and medium (5-8 mm diameter) sized follicles was demonstrated. Next, the effect of E2 during temporary inhibition of GVBD by ROS on the subsequent nuclear maturation was evaluated. Oocytes from small and medium sized follicles were cultured in the presence of ROS, FSH and with or without E2 for 24 h. After this period, oocytes were cultured for another 24 h with FSH but without ROS and E2, after which the nuclear stages and the developmental competence of oocytes were assessed. In conclusion, it is demonstrated that exposure to E2, during temporary inhibition of the GVBD with ROS, affected neither nuclear nor cytoplasmic maturation of oocytes originating from small and medium sized follicles. It might be that in vivo, the increase of E2 during follicle growth is more related to selection of the dominant follicle than to the cytoplamsic maturation of the oocyte as such.

Differential testosterone response to GnRH-induced LH release before and after musth in adult Asian elephant (Elephas maximus) bulls.

Bull elephants exhibit marked increases in testosterone secretion during musth, and studies have shown a heightened sensitivity of the testis to GnRH-stimulated testosterone production in musth compared to nonmusth males. However, activity of the hypothalamo-pituitary-gonadal axis before or soon after musth has not been studied in detail. The aim of this study was to evaluate LH and testosterone responses to GnRH challenge in nine adult Asian elephant (Elephas maximus) bulls during three periods relative to musth: premusth, postmusth, and nonmusth. Bulls were administered 80 µg of a GnRH agonist, and blood was collected before and after injection to monitor serum hormone concentrations. The same bulls were injected with saline 2 weeks before each GnRH challenge and monitored using the same blood collection protocol. All bulls responded to GnRH, but not saline, with an increase in LH and testosterone during all three periods. The mean peak LH (1.76 ± 0.19 ng/mL; P < 0.001) and testosterone (6.71 ± 1.62 ng/mL; P = 0.019) concentrations after GnRH were higher than the respective baselines (0.57 ± 0.07 ng/mL, 3.05 ± 0.60 ng/mL). Although basal- and GnRH-induced LH secretion were similar across the stages, evaluation of the area under the curve in GnRH-treated bulls indicated that the testosterone response was greatest during premusth (2.84 ± 0.76 area units; P = 0.019) compared to postmusth (2.02 ± 0.63 area units), and nonmusth (2.01 ± 0.46 area units). This confirms earlier reports that GnRH stimulates LH release and subsequent testosterone production in bull elephants. Furthermore, although the hypothalamo-pituitary-gonadal axis is active throughout the year, the testis appears to be more responsive to LH in terms of testosterone production in the period leading up to musth, compared to the nonmusth and postmusth periods. This heightened sensitivity, perhaps as a result of LH receptor up-regulation, may prime the testis for maximal testosterone production, leading to the physiological and behavioral changes associated with musth.

Potential of a gonadotropin-releasing hormone vaccine to suppress musth in captive male Asian elephants (Elephas maximus).

Musth in adult bull elephants is a period of increased androgen concentrations ranging from a few weeks to several months. For captive elephant bull management, musth presents a serious challenge because of the aggressive behavior of musth bulls toward people and other elephants. Commercially available GnRH vaccines have been shown to suppress testicular function by interrupting the hypothalamo-pituitary-gonadal (HPG) axis in many species. The aim of this study was to test the efficacy of a GnRH vaccine in elephant bulls for suppressing the HPG axis and mitigating musth-related aggressive behavior. Five adult Asian elephant bulls (22-55 years old) were immunized with a GnRH vaccine starting with an initial injection 2-4 months before the predicted musth period, and followed by three boosters at approximately 4-week intervals. Blood samples were collected twice weekly for hormone and antibody titer analysis. An increase in GnRH antibody titers was observed in all bulls after the second or third booster, and titers remained elevated for 2-3 months after the final booster. Musth was attenuated and shortened in three bulls and postponed completely in two. We conclude that GnRH vaccination is capable of suppressing symptoms of musth in adult bull elephants. With appropriate timing, GnRH vaccination could be used to control or manage musth and aggressive behavior in captive elephant bulls. However, more work is needed to identify an optimal dose, booster interval, and vaccination schedule for complete suppression of testicular steroidogenesis.

Effect of caspase inhibitors on the post-thaw motility, and integrity of acrosome and plasma membrane of cryopreserved equine spermatozoa.

The present study was designed to test the hypothesis that addition of anticaspase cocktails (inhibiting caspases and thus blocking apoptosis) to the extenders increases the post-thaw viability of equine spermatozoa. The addition of caspase inhibitors failed to improve the acrosome and plasma membrane integrity of spermatozoa, suggesting that in equine sperm cryopreservation protocols, the addition of these caspase inhibitors to cryopreservation medium may not be beneficial in protecting the sperm from the stress of cryopreservation.

Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes.

Follicular fluid from 2 to 4 and 5 to 8 mm diameter non-atretic follicles (SFF and LFF, respectively) of sows was added during IVM of cumulus oocytes complexes (COCs) to study its effects on cumulus expansion, nuclear maturation, and subsequent fertilization and embryo development in presence or absence of recombinant human FSH. COCs aspirated from 2 to 5 mm follicles of sow ovaries, were cultured for the first 22 h in TCM-199 and 100 microM cysteamine, with or without 10% pFF and/or 0.05 IU/ml recombinant hFSH. For the next 22 h, the COCs were cultured in the same medium, but without pFF and FSH. After culture, cumulus cells were removed and the oocytes were either fixed and stained to evaluate nuclear stages or co-incubated with fresh sperm. Twenty-four hours after fertilization, presumptive zygotes were fixed to examine fertilization or cultured for 6 days to allow blastocyst formation. Subsequently, embryos were evaluated and the blastocysts were fixed and stained to determine cell numbers. When LFF was added to maturation medium, cumulus expansion and percentage of nuclear maturation (277 +/- 61 microm and 72%, respectively) of COCs were significantly higher (P < 0.05) than those in SFF (238 +/- 33 microm and 55%, respectively). However, in the presence of FSH both FF stimulated cumulus expansion and nuclear maturation to a similar degree. No differences were observed with regards to sperm penetration, male pronucleus formation, and to polyspermia between fertilized oocytes matured either in SFF or LFF. Fertilized oocytes matured in the presence of LFF without or with FSH showed a higher cleavage (45 +/- 7% and 51 +/- 7%, respectively) and blastocyst (14 +/- 4% and 22 +/- 6%, respectively) formation rate compared to SFF (cleavage, 35 +/- 8% and 41 +/- 4%, blastocyst: 8 +/- 3 and 13 +/-3, respectively; P < 0.05). The mean number of cells per blastocyst did not differ significantly between treatments. These findings indicate that factor(s) within follicles at later stages of development play an important role during oocyte maturation and thereby enhance developmental competence to occur.