Proteomic Analysis of Domestic Cat Blastocysts and Their Secretome Produced in an In Vitro Culture System without the Presence of the Zona Pellucida.

Domestic cat blastocysts cultured without the zona pellucida exhibit reduced implantation capacity. However, the protein expression profile has not been evaluated in these embryos. The objective of this study was to evaluate the protein expression profile of domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were generated: (1) domestic cat embryos generated by IVF and cultured in vitro (zona intact, (ZI)) and (2) domestic cat embryos cultured in vitro without the zona pellucida (zona-free (ZF group)). The cleavage, morula, and blastocyst rates were estimated at days 2, 5 and 7, respectively. Day 7 blastocysts and their culture media were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The UniProt Felis catus database was used to identify the standard proteome. No significant differences were found in the cleavage, morula, or blastocyst rates between the ZI and ZF groups (p > 0.05). Proteomic analysis revealed 22 upregulated and 20 downregulated proteins in the ZF blastocysts. Furthermore, 14 proteins involved in embryo development and implantation were present exclusively in the culture medium of the ZI blastocysts. In conclusion, embryo culture without the zona pellucida did not affect in vitro development, but altered the protein expression profile and release of domestic cat blastocysts.

A Subovulatory Dose of Human Chorionic Gonadotropin (hCG) May Sustain Terminal Follicle Development and Reproductive Efficiency during Anestrus in Sheep.

The study tested the hypothesis that a single administration of hCG supports the LH-dependent phase of terminal follicular development in synchronized sheep during anestrus, using eCG as a functional reference. Using a clinical approach, four experiments were designed to achieve the following: (1) Identify the inhibitory influence of anestrus on reproduction efficiency; (2) Assess the potential of hCG to keep functional blood concentrations after a single dose; (3) Characterize the effect of different doses of hCG on reproductive functional markers; (4) To compare the ability of hCG to that of eCG to support follicular development and fertility based on the same markers. The results showed that anestrus seems to affect follicular and luteal function under LH dependency as FSH-dependent markers are not compromised; hCG maintains higher blood concentrations than controls for at least 48 h; hCG improves follicular development and ovulatory rates compared to controls and at standards comparable to a breeding season; and ewes treated with hCG exhibit similar performance to those treated with eCG. Our results conclude that hCG can be used to support follicular function during anestrus in sheep, aiming to perfect its regulation in assisted reproduction.

Unveiling the Role of IGF-I in Fertility: Effect of Long-Acting Bovine Somatotropin (bST) on Terminal Follicular Development and Fertility during an Annual Reproductive Cycle in Sheep.

The study aimed to assess the effect of long-acting bST treatment, in a dose that only increases IGF-I plasma concentrations, on ovarian and fertility markers of estrous synchronized ewes that were fed to keep their bodyweight. Three experiments were designed to evaluate this effect: in Experiment 1, 18 ewes were distributed in groups (bST 0, 30, 50 mg) to measure plasma IGF-I and insulin for 15 days; in Experiment 2, 92 ewes (5 replicates) in two groups (0 and 30 mg bST) were synchronized using a 6-day progesterone protocol during the breeding season to assess the effect of bST on follicular and luteal performances, estrous and ovulation, and fertility after mating. In Experiment 3, 50 ewes (3 replicates) were used to repeat the study before but during anestrus. Results indicate that 50 mg bST increased IGF-I and insulin plasma concentrations, but 30 mg bST only increased IGF-I concentrations; and that only during the breeding season did 30 mg bST increase the number of lambs born and the reproductive success of ovulatory-sized follicles compared to controls. This occurred without it affecting any other reproductive marker. In conclusion, 30 mg bST treatment may improve oocyte competence for fertility during the breeding season.

Zona pellucida removal modifies the expression and release of specific microRNAs in domestic cat blastocysts.

The in vitro culture of domestic cat embryos without the zona pellucida affects their implantation capacity. MicroRNAs (miRNAs) have an important role in embryo-maternal communication and implantation. The objective of this study was to evaluate the expression of specific miRNAs in domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were done: (1) domestic cat embryos cultured with the zona pellucida (zona intact control group, ZI); and (2) cultured without the zona pellucida (zona free group, ZF). The cleavage, morula and blastocyst rates were evaluated. The blastocysts and their spent medium were used for miRNA expression analysis using RT-qPCR (miR-21, miR-24, mi25, miR-29, miR-96, miR-98, miR-103, miR-191, miR-196, miR-199, miR-130, miR-155 and miR-302). The pre-mature microRNAs (pre-miRNAs) and miRNAs were evaluated in the blastocysts and only miRNAs were evaluated in the spent medium. No differences were observed in the cleavage, morula and blastocyst rates between the ZF and ZI groups (P > 0.05). For miRNAs analysis, miR-103 and miR-191 had the most stable expression and were selected as internal controls. ZF blastocysts had a higher expression of miR-21, miR-25, miR-29 and miR-199 and a lower expression of miR-96 than their ZI counterparts (P < 0.05). Furthermore, higher levels of miR-21, miR-25 and miR-98 were detected in the spent medium of ZF blastocysts (P < 0.05). In conclusion, in vitro culture of domestic cat embryos without the zona pellucida modifies the expression of miR-21, miR-25, miR-29, miR-199 and miR-96 at the blastocyst stage and the release of miR-21, miR-25 and miR-98.

First molecular detection of Equine Herpesvirus type 3 (EHV-3) in Chile.

Equine coital rash (ECE) is a highly contagious benign infection that induces lesions on external genitals, and it is caused by the equine herpesvirus type 3 (EHV-3). Although the disease is globally distributed, its presence in Chile has not been documented from a genetic point of view. Here, we performed polymerase chain reaction screenings for EHV-3 in lesions of external genitals in four horses belonging to a riding station at Bulnes, Ñuble Region, Chile. We sequenced a fragment of the glycoprotein G (gG) gene from three horses with clinical signs of ECE. The sequences were identical between them and 99.7% similar to a haplotype of EHV-3 detected in Brazil, and phylogenetically related with homologue from Japan, Russia and Brazil. Our results show the presence of EHV-3 for the first time in horses with ECE in Chile.

Analysis of trophectoderm markers in domestic cat blastocysts cultured without zona pellucida.

Domestic cat embryos generated by in vitro fertilization (IVF) and cultured without the zona pellucida have a reduced implantation capacity after embryo transfer at the blastocyst stage. The objective of this study was to evaluate the expression of trophectoderm markers in domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were selected: (1) domestic cat embryos generated by IVF and cultured in vitro normally (zona intact group, ZI); and (2) domestic cat embryos generated by IVF and cultured in vitro without a zona pellucida (zona-free group, ZF). In the ZF group, the zona pellucida of the presumptive zygote was removed and these were cultured using the well of the well (WOW) system. In vitro culture was carried out for 7 days. The cleavage, morula and blastocyst rates were estimated. Finally, the relative expression levels of the trophectoderm markers TEAD4, YAP1, CDX2 and EOMES, the cell adhesion marker E-cadherin and the apoptosis marker CASP3 were evaluated by RT-qPCR in the blastocysts. The Wilcoxon test was used to evaluate differences (P < 0.05). No differences were observed in the cleavage, morula and blastocyst rates between the ZF and ZI groups. No differences were found in the expression of TEAD4, CDX2, E-cadherin and CASP3 between groups. The expression of YAP1 and EOMES was higher in ZF blastocysts than in ZI blastocysts. In conclusion, the in vitro culture without the zona pellucida generates an overexpression of YAP1 and EOMES in the domestic cat blastocysts. More studies are needed to confirm if this overexpression might affect in vivo development.

Domestic cat embryos generated without zona pellucida are capable of developing in vitro but exhibit abnormal gene expression and a decreased implantation rate.

The removal of the zona pellucida has been used to improve the in vitro development of domestic cat embryos generated by IVF and SCNT. However, the in vivo development of domestic cat embryos generated without the zona pellucida has not been evaluated. The objective of this study was to evaluate the effects of zona pellucida removal on the in vitro and in vivo development of domestic cat embryos generated by IVF. For this purpose, two experimental groups were created: 1) domestic cat embryos cultured in vitro (Zona-intact group, ZI) and 2) domestic cat embryos cultured in vitro without the zona pellucida (Zona-free group, ZF). Domestic cat embryos were generated by IVF and cultured in vitro for 8 days. In the ZF group, the zona pellucida was removed after IVF, and embryos were cultured using the well of the well system (WOW). Cleavage, morula and blastocyst rates were evaluated in both groups. The diameter and total cell number of blastocysts were assessed. Relative expression of pluripotency (OCT4, SOX2 and NANOG), differentiation (CDX2 and GATA6) and apoptotic markers (BAX and BCL2) was evaluated in blastocysts. Finally, to evaluate in vivo development, embryos at days 5, 6 and 7 of development were transferred into recipient domestic cats, and ultrasonography was performed to evaluate implantation. No differences were observed in the cleavage, morula or blastocyst rates between embryos from the ZI and ZF groups. The diameter (mean ± SD) of blastocysts from the ZF group was greater (253.4 ± 83.3 µm) than that from the ZI group (210.5 ± 78.5 µm). No differences were observed in the relative expression of OCT4, CDX2 or GATA6. However, the relative expression of SOX2 and NANOG was significantly reduced in ZF blastocysts compared to ZI blastocysts. Furthermore, the relative expression of BAX was higher in ZF blastocysts than in ZI blastocysts. Finally, four pregnancies were confirmed after the transfer of ZI embryos (n = 110). However, no pregnancies were observed after the transfer of ZF embryos at the morula or blastocyst stage (n = 56). In conclusion, domestic cat embryos cultured without the zona pellucida were able to develop in vitro until the blastocyst stage. However, the removal of the zona pellucida negatively affected the gene expression of pluripotency and apoptosis markers, and ZF embryos were unable to implant. This might indicate that the removal of the zona pellucida is detrimental for the implantation and in vivo development of domestic cat embryos.

Distinctive Cellular Transcriptomic Signature and MicroRNA Cargo of Extracellular Vesicles of Horse Adipose and Endometrial Mesenchymal Stem Cells from the Same Donors.

Equine endometrial and adipose mesenchymal stem cells (eMSCs and aMSCs, respectively) were isolated from the same donors of thoroughbred mares. The cells displayed characteristic features of MSCs, including trilineage mesodermal and also neurogenic differentiation. We evaluated the influence of cellular origin on their transcriptome profile. Cellular RNA was isolated and sequenced and extracellular vesicles (EVs) were obtained from conditioned medium of cells cultured in medium depleted of EVs, and their microRNA (miRNA) cargo analyzed by sequencing. Differential expression of mRNAs and EV-miRNA was analyzed, as well as pathways and processes most represented in each cell origin. mRNA reads from all expressed genes clustered according to the cellular origin. A total of 125 up- and 51 downregulated genes were identified and 31 differentially expressed miRNAs. Based on mRNA sequencing, endometrial MSCs strongly upregulated genes involved in the Hippo, transforming growth factor beta, and pluripotency signaling pathways. Alongside with this, pathways involved in extracellular matrix reorganization were the most represented in the miRNA cargo of EVs secreted by eMSCs. The niche from which MSCs originated defined the transcriptomic signature of the cells, including the secretion of lineage-specific loaded EV to ensure proper communication and homeostasis. Identification and testing their biological functions can provide new tools for the therapeutic use of horse MSC.

Assessment of the anti-inflammatory and engraftment potential of horse endometrial and adipose mesenchymal stem cells in an in vivo model of post breeding induced endometritis.

Horse mesenchymal stem cells (MSC) are potential anti-inflammatory tools for post-breeding induced endometritis (PBIE). In this research MSCs isolated from the endometrium or subcutaneous fat of the same donors were infused iu into mares with PBIE for assessment of their anti-inflammatory action and engraftment. PBIE was induced in nine gynecologically healthy mares by iu infusion of 500 million dead sperm in saline. Inflammatory markers were analyzed in uterine lavages and biopsies immediately before (phase I) and 3 h after infusion of sperm (phase II). Measurements: polymorph nuclear cells (PMN), proteins IL-6 and TNFα (ELISA in the lavages) and immunostaining in biopsies, transcripts of IL-1α, 6, 8, 10, TNFα and COX2 (qPCR of pelleted lavages). At 24 h after sperm deposition (phase III), mares were instilled iu with 20 ml of saline containing 2 × 107 adipose MSCs (n = 3, group 1) or endometrial MSCs (n = 3, group 2). Cells were labeled previously with carboxyfluorescein diacetate succinimidyl ester (CFDA SE). A third group (n = 3) received 20 mL of sterile saline alone. After 48 h another biopsy/lavage were done and the same parameters analyzed. For engraftment, additional biopsies were taken at days 10 and 30 of sperm infusion and analyzed by confocal microscopy. Dead sperm in saline markedly increased PMNs counts, IL-6 and TNFα expression in the ELISA (p < 0.05) and immunostaining. In phase III a significant reduction (p < 0.0001) of PMN was found in all samples, including control mares. A decrease (p < 0.05) of IL-6 and TNF-α was detected by ELISA, in the groups that received MSC, but not in control group. In the aMSC-treated group, a significant decrease was found in the expression of (IL1α, p = 0.0003; IL-6 p 0.04; IL-8, p = 0.006, TNFα p = 0.004). Expression of IL-10 and COX2 remained unchanged (p = 0.08). In the mares that received eMSC, IL-6 and 8 decreased significantly (p = 0.01), IL-10 increased (p = 0.009), while TNFα, COX2 and IL1α did not significantly change their expression. In the engraftment experiment CFDA label was found sparingly in all the samples analyzed until day 30, mainly at the stromal compartment of the endometrium. No differences in the engraftment pattern was found among cell origins. We conclude that inoculation of MSCs significantly reduced inflammation independently of the origin of the cells and that cells perform limited engraftment detectable after one month of infusion. These findings can be of help for the design of new anti-inflammatory therapies of uterine diseases in mares.

Endometrial Stem Cells in Farm Animals: Potential Role in Uterine Physiology and Pathology.

The endometrium is an accessible source of mesenchymal stem cells. Most investigations of endometrial mesenchymal stem cells (eMSCs) have been conducted in humans. In animals, particularly in livestock, eMSC research is scarce. Such cells have been described in the bovine, ovine, caprine, porcine, and equine endometrium. Here we provide the state of the art of eMSCs in farm animals with a focus on the bovine species. In bovines, eMSCs have been identified during the phases of the estrous cycle, during which their functionality and the presence of eMSC-specific markers has been shown to change. Moreover, postpartum inflammation related to endometritis affects the presence and functionality of eMSCs, and prostaglandin E2 (PGE2) may be the mediator of such changes. We demonstrated that exposure to PGE2 in vitro modifies the transcriptomic profile of eMSCs, showing its potential role in the fate of stem cell activation, migration, and homing during pathological uterine inflammation in endometritis and in healthy puerperal endometrium. Farm animal research on eMSCs can be of great value in translational research for certain uterine pathologies and for immunomodulation of local responses to pathogens, hormones, and other substances. Further research is necessary in areas such as in vivo location of the niches and their immunomodulatory and anti-infective properties.

Endometritis and In Vitro PGE2 Challenge Modify Properties of Cattle Endometrial Mesenchymal Stem Cells and Their Transcriptomic Profile.

Mesenchymal stem cells (MSCs) were isolated and characterized from postpartum bovine endometrium of animals with subclinical (n = 5) and clinical endometritis (n = 3) and healthy puerperal females (n = 5). Cells isolated displayed mean morphological features of MSCs and underwent osteogenic, chondrogenic, and adipogenic differentiation after induction (healthy and subclinical). Cells from cows with clinical endometritis did not undergo adipogenic differentiation. All cells expressed mRNAs for selected MSC markers. Endometrial MSCs were challenged in vitro with PGE2 at concentrations of 0, 1, 3, and 10 µM, and their global transcriptomic profile was studied. Overall, 1127 genes were differentially expressed between unchallenged cells and cells treated with PGE2 at all concentrations (763 up- and 364 downregulated, fold change > 2, and P < 0.05). The pathways affected the most by the PGE2 challenge were immune response, angiogenesis, and cell proliferation. In conclusion, we demonstrated that healthy puerperal bovine endometrium contains MSCs and that endometritis modifies and limits some functional characteristics of these cells, such as their ability to proceed to adipogenic differentiation. Also, PGE2, an inflammatory mediator of endometritis, modifies the transcriptomic profile of endometrial MSCs. A similar situation may occur during inflammation associated with endometritis, therefore affecting the main properties of endometrial MSCs.

The vanguard sperm cohort of the boar ejaculate is overrepresented in the tubal sperm reservoir in vivo.

In boars, sperm cohorts are sequentially emitted in epididymal cauda fluid and resuspended in different mixtures of accessory sex gland secretions while ejaculated in various fractions. During natural mating, these ejaculate fractions sequentially enter the cervix-uterine lumen, are quickly transported towards the tips of the uterine horns and colonize the oviductal sperm reservoirs (SR). Using a simple experiment, we tested the hypothesis that the first ejaculated sperm subpopulation (fortuitously present in the peak portion of the sperm-rich fraction [SRF], the so-called Portion 1, P1) is, by reaching first the SR, overrepresented there. Spermatozoa from P1- and of P2- (last portion of the SRF and the Post-SRF) were collected from 3 fertile boars. P1-spermatozoa were fluorophore DNA-stained, while P2-spermatozoa were kept unstained. Weaned estrous sows were conventionally inseminated (12 h after onset of estrus) with similar sperm numbers (approx 10 x 10(9) spermatozoa) per portion but in different orders as follows: (i) a mix of P1 and P2 aliquots (control, P1+P2, n=5), or testing (ii) a sequential order (P1-P2, Treatment A, n=5) or (iii) an inverse order (P2-P1, Treatment B, n=5) of cohort AI. Sows were euthanized approx 3 h post-AI, and the SRs were flushed to recover the spermatozoa, which were accounted for as stained or unstained. The total number of spermatozoa flushed did not differ between groups or boars (NS, ranging 0.9 to 2.0 x 10(9)). Sequential, in vivo-like, sperm deposition (P1-P2, Treatment A) yielded the highest proportion of stained P1-spermatozoa in the SRs (59.8 +/- 5.66%, means +/- SEM) compared with when the order was reversed (P2-P1, Treatment B; 15.6 +/- 2.1% P1-spermatozoa, P<0.05) or P1 and P2 sperm suspensions were mixed (control, 36.9 +/- 2.70% P1-spermatozoa, P<0.05). The tested hypothesis proved valid; if inseminated in the same order as ejaculated in vivo, P1-spermatozoa become overrepresented in the SR. The physiological consequences of this skewed SR-colonization are discussed in this paper alongside the advantageous use of P1-spermatozoa for handling, including cryopreservation.

Selection of boar spermatozoa using centrifugation on a glycidoxypropyltrimethoxysilane-coated silica colloid.

Use of sperm separation methods such as density gradient centrifugation for selecting the best spermatozoa for animal breeding is constrained by the problem of dealing with the large volumes of ejaculate produced by the males of some species, such as boars. The purpose of this study was to compare density gradient centrifugation (DGC) with centrifugation on a single layer of colloid (SLC) for the preparation of ejaculated boar spermatozoa using Androcoll(TM)-P. There was no difference between the two techniques in terms of sperm motility or duration of motility after selection, and sperm motility was retained for at least 24 h longer in the centrifuged sperm preparations than in controls (uncentrifuged aliquots). Sperm motility was significantly better (P<0.001) in the centrifuged sperm preparations (means +/- sd: SLC 79.6 +/- 8.1% and DGC 74.2 +/- 12.0%) than in the uncentrifuged controls (62.9 +/- 12.7%). The mean yield of motile spermatozoa for SLC was 67.5 +/- 25.6%, and for DGC it was 59.6% +/- 22.3% (not significant, ns). Sperm survival was significantly increased by colloid centrifugation (control 3.1 +/- 0.3 days, SLC 5.5 +/- 0.79 days, DGC 5.75 +/- 0.62 days; P<0.001 for uncentrifuged versus centrifuged; SLC vs. DGC, ns). Moreover, boar spermatozoa could be stored for 24 h before centrifugation without any detrimental effect on sperm motility or duration of motility. In a further experiment, larger volumes of ejaculate were processed easily on a modified SLC, indicating that this method may be practical for processing large volumes of boar ejaculates.

Cryo-scanning electron microscopy (Cryo-SEM) of boar semen frozen in medium-straws and MiniFlatPacks.

In this study we demonstrate, in the frozen state, the architecture of frozen boar spermatozoa collected from the sperm-rich fraction of ejaculates (n=13) from four fertile boars packed and split-frozen in medium-straws (MS) and MiniFlatPacks (MFP), cross-sectioned in the frozen state and evaluated by image analysis on images obtained by use of cryo-scanning electron microscopy (Cryo-SEM). The tested hypothesis was that the degree of in situ dehydration and levels of homogeneity of boar semen either frozen in MSs or MFPs packages differ between them, with MFPs allowing for a more uniform dehydration of the spermatozoa and a higher cryosurvival, monitored by computer assisted sperm analysis (CASA) as proportion of linearly motile spermatozoa, compared to semen packaged and processed in MSs. The organization and relative surface of biological material (veins; e.g., frozen extender, bound water, solutes and spermatozoa) as well as free water (lakes) was measured as the degree of dehydration of the samples. The apparent organization of lakes and veins differed between packages, with the MFPs depicting larger lakes than the MSs. The sizes of the lakes in the latter appeared, moreover, highly asymmetrical depending on their position of the section. The relative surface of these lakes per section, respectively veins differed between packages (P<0.05), indicating a larger amount of free-water (lakes; 81.73+/-2.07% vs. 77.91+/-1.57%) in the MFPs and, consequently, thinner veins than in MSs. In conclusion, MFPs seem to allow for a more homogenous dehydration of the spermatozoa/frozen extender compared to MSs, which might account for their somewhat better sperm quality post-thaw.

Differences in SCSA outcome among boars with different sperm freezability.

Spermatozoa from some boars sustain the process of cryopreservation poorly and yield poor fertility after artificial insemination. Poor freezability has not been disclosed using conventional semen analyses. A defective chromatin can, if present in a substantial number of spermatozoa, affect the fertilizing ability of spermatozoa. Here we tested the hypothesis that nuclear DNA instability could explain differences in freezability among boars, and complement or supersede conventional tests for sperm quality such as sperm motility or membrane assessments. Frozen-thawed (FT) spermatozoa from a total of 44 stud boars were assessed by the sperm chromatin structure assay (SCSA), in relation to computer-assisted sperm analysis-derived sperm motility variables and sperm viability (triple fluorescent microscopic staining), including three experiments. The first trial, including 24 boars, evaluated the relationship between the sperm motility and viability with levels of DNA integrity. The SCSA showed that most spermatozoa had intact DNA [levels of DNA fragmentation index (%DFI) ranging from 0.63% to 11.85%] significantly correlated (albeit weakly) with current sperm quality variables. The second trial, on 15 boars, assessed the influence of two different thawing rates (20 s at 37 degrees C vs. 8 s at 70 degrees C) and the post-thaw incubation times (0, 30, 150 and 300 min) at 37 degrees C on FT-boar sperm quality. The highest sperm survival (p < 0.05) and the lowest DNA damage (p < 0.01) were achieved when thawing was carried out at 70 degrees C for 8 s, without any change during the first 150 min of incubation. Finally, the third experiment studied if differences in sperm freezability showed by stud boar semen, as 'good' or 'bad' freezers by conventional analyses, could be attributed to differences in chromatin structure. All SCSA parameters were low, but significantly (p < 0.05-0.001) higher for 'bad' freezers, showing they had less homogeneous sperm chromatin than the 'good' freezers. The results indicate that SCSA outcome complements conventional assessment of FT-boar spermatozoa, disclosing differences in their ability to sustain freezing and thawing. However, the low overall DNA damage observed in FT spermatozoa seems to have poor biological significance.

Effect of storage in short--and long-term commercial semen extenders on the motility, plasma membrane and chromatin integrity of boar spermatozoa.

For artificial insemination (AI) in pigs, preservation of liquid boar semen at 16-20 degrees C is still common practice as sperm cryopreservation remains suboptimal in this species. To meet the different needs of the swine industry, several extenders have been developed to preserve semen in liquid form for short--and long-term storage. In the present study, three different commercial extenders devised for short-term (BTS+) or long-term preservation (MR-A and X-Cell), were used to test whether storage of semen from four mature, fertile boars at 17 degrees C for 96 h would affect sperm characteristics relevant for fertility, such as motility, membrane integrity and chromatin stability. Computer-assisted sperm analysis, and stainings with the acylated membrane dye SYBR-14/propidium iodide, and acridine orange in connection with flow cytometry were used to evaluate these variables. Percentages of total motile spermatozoa decreased slightly, but significantly, after 72-96 h. While membrane integrity values varied during the period of study, no significant changes in either membrane integrity or chromatin stability were, however, registered. This suggests a customary 96-day storage at 17 degrees C in these extenders was too short an interval to cause losses of integrity in nuclear DNA in the boar population studied.

Does exposure to di(2-ethylhexyl) phthalate in pre-pubertal boars affect semen quality post-puberty?

Di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in polyvinyl chloride (PVC) products, has been ascribed to have toxic effects on animal reproduction. The present study aimed at determining potential late effects of pre-pubertal oral exposure to DEHP on semen quality in young pigs. Ten pairs of cross-bred male siblings were used. One brother in each pair became, at random, the test animal while the other acted as control. Test males were exposed to 300 mg/kg body weight (bw) of DEHP administered orally three times a week from 3 to 7 weeks of age. The control group was given placebo (water). Semen analyses started when the boars reached 6 months of age, with semen collected twice weekly, until animals were 9 months of age. Semen was evaluated for ejaculate volume, sperm concentration, total sperm count, sperm motility, sperm morphology (including presence of cells other than spermatozoa) and sperm plasma membrane integrity. Total sperm motility tended to be lower while local motility was higher in the DEHP-exposed group compared with controls (p = 0.07) when assessed by computer-assisted sperm analysis. The DEHP-exposed group had a significantly (p < 0.05) lower percentage of spermatozoa with tailless, defective heads (at 7-8 months of age) and double-folded tails (at 6-7, 7-8 and 6-9 months of age), compared with controls (albeit always under 5%). In summary, there were no obvious adverse effects of early oral exposure to 300 mg/kg bw of DEHP on sperm output and sperm quality in post-pubertal young boars.

Fertility after deep intra-uterine artificial insemination of concentrated low-volume boar semen doses.

UNLABELLED: Boar semen can be successfully frozen - highly packed - in small containers (medium-straw, MS or MiniFlatPack, MFP). The use of deep intra-uterine artificial insemination (DIU-AI) can make possible the deposition of small volumes of this thawed, non re-extended semen deeply intra-uterine, close to the sperm reservoir. The present experiments studied the fertility achieved after single or double DIU-AI per oestrus, with special attention to the interval between AI and spontaneous ovulation. Semen from two boars of proven fertility was frozen in MS or MFP holding 1 x 10(9) total spermatozoa. Multiparous (2-5 parity, n=42) crossbred sows were checked for oestrous behaviour after weaning and the occurrence of spontaneous ovulation was checked with transrectal ultrasonography (TUS) to establish the mean interval between onset of oestrus (OO) and ovulation which was found to be when approximately 2/3 of the oestrus period has passed. The sows were, in the following standing oestrus, subjected to DIU-AI using thawed semen from either MS (n=20) or MFP (n=22), inseminated without further re-extension. The sows were randomly allotted to one of three groups: (1) single DIU-AI 8 h before expected ovulation (control group, n=19); (2) single DIU-AI 4 h before expected ovulation (treatment group S, n=15); and (3) double DIU-AI 12 and 4 h before expected ovulation (treatment group D, n=8). Occurrence of spontaneous ovulation was confirmed by TUS, performed as during the first oestrous period and used to determine the real interval of DIU-AI and ovulation. Pregnancy was also confirmed by TUS 28 days after OO in those sows not returning to oestrus. These sows were slaughtered (30-45 days of pregnancy), and the appearance of the reproductive tract and ovaries, the number of live and dead foetuses, of implantation sites and of corpora lutea (CL) were recorded. Sows (n=9) returning to oestrus ("open") were re-inseminated (either once [n=4] or twice [n=5]) the following oestrus with either MFP (n=5) or MS (n=4) and slaughtered 12-14 h post-ovulation for recovery of tubal oocytes and of spermatozoa from the uterotubal junctions (sperm reservoir), to assess the degree of effectiveness of sperm transport. Post-thaw sperm motility was 44.3+/-3.21% in MFP and 42.8+/-0.72% for MS (LSmean+/-S.E.M., n.s.), and did not significantly change from thawing to AI. The DIU-AI could be performed in all sows, but insertion was difficult (slow >5 min) in 5/42 sows. Four of these sows returned to oestrus. Pregnancy rate averaged 35% (group D: 25%, group S: 40%, control: 36%, n.s.). The interval between DIU-AIs and spontaneous ovulation varied largely, ranging from -13 to -3 h for group C, for group S from -11 to +3 h and for group D from -17 to -4 h. Pregnancy rates were clearly related to the interval DIU-AI and ovulation, being highest (60%, 12/20) when AI occurred between 8 and 4 h before spontaneous (not expected) ovulation. The number of implantation sites ranged 6-22 (n.s. among groups), and the number of alive foetuses 2-11 (n.s. among groups). Implantation rate (total number of implantations/CL) ranged 48.0-69.7% being highest in the D-group (P<0.05). The examination of the "open" sows slaughtered 12-14 h post-ovulation revealed few recovered oocytes were fertilized (approximately 10%). Only 40% of oocytes had spermatozoa bound to the zona pellucida, not more than two spermatozoa per oocyte. Moreover, low sperm numbers (approximately 4000) were found in the sperm reservoirs (UTJs), irrespective of using single or double DIU-AI (n.s.). The highest values (P<0.05) for these variables were recorded when DIU-AI (either single or double [second AI]) occurred 4-8 h before ovulation, especially when MFP-semen was used (P<0.05). IN CONCLUSION: (1) DIU-AI can be easily performed in most sows; (2) pregnancies can be obtained by the DIU-AI of low volumes of highly concentrated frozen-thawed boar semen, once or twice during oestrus, but fertility is still low, probably owing to an unsatisfactory sperm transport when expected and real ovulation differ; and (3) fertility is related to the interval DIU-AI and ovulation which should be -8 to -4 h of spontaneous ovulation and to the package, MFP having shown better results in vivo. The results stress the need for careful, and frequent, control of oestrus signs.

Identification of sperm morphometric subpopulations in two different portions of the boar ejaculate and its relation to postthaw quality.

A statistical approach using sequentially principal component analysis (PCA), clustering, and discriminant analyses was developed to identify sperm morphometric subpopulations in well-defined portions of the fresh boar ejaculate. Semen was obtained as 2 portions (the first 10 mL of the sperm-rich fraction and the rest of the ejaculate, respectively) and frozen using a conventional protocol. Before freezing, an aliquot was used for computer-assisted sperm morphometry analysis (ASMA). Postthaw quality was evaluated using computer-assisted sperm analysis (CASA), and an annexin-V/PI assay evaluated sperm membranes. The PCA revealed that 3 variables represented more than 78% of the cumulative variance in sperm subpopulations. The clustering and discriminant analyses, based on 5780 individual spermatozoa, revealed the existence of 4 sperm subpopulations. The relative percentage of these subpopulations varied between boar and ejaculate portions. Linear regression models based on measured morphometric characteristics could account for up to 36% of the percentage of intact sperm membranes postthaw. The ASMA protocol used in our study was useful to detect subtle morphometric differences between spermatozoa, and the combination of this analysis with a multivariate statistical procedure gave new information on the biological characteristics of boar ejaculates that is not given by conventional sperm analysis.

Pregnancy rates in repeat-breeder heifers following multiple artificial inseminations during spontaneous oestrus.

Hormonal asynchronies during oestrus, related to the presence of suprabasal plasma-progesterone (P4) concentrations and a delayed ovulation, interfere with the fertility of repeat-breeder heifers (RBH). Since tubal dysfunction can occur in connection with hormonal asynchronies and constrained availability of fertile spermatozoa at the time of ovulation, the present study tested the hypothesis that frequent sperm deposition from onset of oestrus to ovulation may improve pregnancy rates in RBH. Five RBH and five virgin heifers (VH; controls) were repeatedly artificially inseminated (AI) at 6 h intervals from onset of oestrus to spontaneous ovulation. Hormone analyses revealed suprabasal P4 concentrations and a delay in the occurrence of the luteinising hormone (LH) surge, but a normal cortisol profile in RBH. Compared with controls, RBH presented longer interval from onset of oestrus to ovulation, and therefore, received more AIs. Pregnancy rates in RBH reached control levels (60%; NS), indicating that the hypothesis might be correct. Pregnancy rates in VH were below the expected range, presumably attributed to a deleterious influence of the frequent handling. The study suggests that pregnancy rates can be improved in RBH by frequent AI in relation to spontaneous ovulation. However, this practice of repeated manipulations, while seeming not to show adverse effects, lacks practicality for routine use.