Routine Artificial Insemination Research Articles

Uterine tone influences fertility of Merino ewes following laparoscopic artificial insemination

Artificial insemination (AI) plays a critical role in facilitating rapid genetic and production gains within the sheep industry. However, variable rates of AI success remain a concern for the industry and a barrier to adoption. Furthermore, the degree to which female factors influence the success of intrauterine laparoscopic AI rather than natural mating remains unknown. As such, this study investigates the effect of several factors collected during the time of AI, on the success of intrauterine laparoscopic AI. Data was generously donated by artificial breeding companies and stud breeders during routine commercial AI operations. AI data was collected over 3 breeding seasons during commercial AI programs (N = 24 programs) using Merino ewes (N = 24,700). Sire ID (N = 253), time of AI following progesterone removal (approx. 43–59 h post removal), uterine tone and intra-abdominal fat (both scored 1–5) as well as age of the ewe were all recorded at the time of AI. Transcutaneous ultrasound subsequently determined pregnancy rate approximately 55 days post-AI. A multivariate regression analysis was performed and revealed pregnancy success to increase when semen was inseminated into a ewe with a uterine tone score of 4 or 5 (P < 0.001). The remaining factors fell short of significance within the multivariate model. An interclass coefficient variation matrix was also used to determine the proportion of variation contributed to AI success by random factors allocated in the model; site, sire, AI date and breeding season (45.99 %, 29.94 %, 15.15 % and 8.92 %, respectively). These results highlight the influence of uterine tone on ewe fertility following laparoscopic AI, but also that program location and the sire used can further modify this influence on pregnancy rate. These factors must now be considered in combination with semen factors per individual sire used during AI to ascertain the contribution of several factors to the success of laparoscopic AI in Australia.

Impact of litter size on the hematological and iron status of gilts, sows and newborn piglets: a comparative study of domestic pigs and wild boars

BackgroundThe critically low hepatic iron stores of newborn piglets are considered to be a major cause of neonatal iron deficiency in modern breeds of domestic pig (Sus domestica). The main factor believed to contribute to this phenomenon is large litter size, which has been an objective of selective breeding of pigs for decades. As consequence, iron transferred from the pregnant sow has to be distributed among a greater number of fetuses.ResultsHere, we investigated whether litter size influences red blood cell (RBC) indices and iron parameters in Polish Large White (PLW) piglets and gilts. Small and large litters were produced by the transfer of different numbers of embryos, derived from the same superovulated donor females, to recipient gilts. Piglets from large litters obtained following routine artificial insemination were also examined. Our results clearly demonstrated that varying the number of piglets in a litter did not affect the RBC and iron status of 1-day-old piglets, with all showing iron deficiency anemia. In contrast, gilts with small litters displayed higher RBC and iron parameters compared to mothers with large litters. A comparative analysis of the RBC status of wild boars (having less than half as many piglets per litter as domestic pigs) and PLW pigs, demonstrated higher RBC count, hemoglobin level and hematocrit value of both wild boar sows and piglets, even compared to small-litter PLW animals.ConclusionsThese findings provide evidence that RBC and iron status in newborn PLW piglets are not primarily determined by litter size, and indicate the need to study the efficiency of iron transport across the placenta in domestic pig and wild boar females.

Determining the clinical utility of a single pre-breeding examination for predicting subsequent reproductive performance in seasonal pasture-based dairy heifers

Heifers that conceive early in the breeding season are key to the maintenance of a compact calving pattern in seasonal dairy herds. The purpose of this prospective observational study was to determine whether a single pre-breeding examination is useful in predicting future reproductive and calving performance in dairy heifers. This evaluation was based on a matrix of easily measurable variables which could be applied to seasonal, pasture-based dairy heifers. The matrix variables were; body weight (≥55% mature body weight at mating start date), body condition score (≥3.5), and the presence of a corpus luteum. Seasonal, pasture-based dairy heifers (n = 572) on six commercial dairy farms were examined at an average of 28 days prior to the breeding season. At this examination, they were classified as READY (meeting all three of the above criteria), INTERMEDIATE (meeting one or two of the above criteria) or PROBLEM (meeting none of the above criteria) based on the matrix and then exposed to a routine artificial insemination (AI) breeding period followed by bull breeding depending on individual farm management. Subsequent pregnancy status was determined by transrectal ultrasonography initially at 30–60 days in calf (DIC), and confirmed at over 60 DIC. After the breeding season, 519 (96%) of heifers were determined to be pregnant, of which 345 (64%) became pregnant within the first three weeks of the breeding season. Farmers collected calving data; including calving difficulty score, from the heifers the following spring. A logistic regression model was used to analyse the association between the matrix categories and the following outcomes: reproductive (21 day submission rate, 1st AI conception rate, 3 wk and 6 wk in calf rate) and calving (assistance and dystocia). Heifers classified as PROBLEM had significantly lower odds of conceiving to the first AI serve and of becoming pregnant within the first three and six weeks of the breeding season (P ≤ 0.05). In conclusion, the proposed matrix could be practically useful for classifying heifers prior to the breeding season.

Improvement of in vitro fertilization by a tannin rich vegetal extract addition to frozen thawed boar sperm

Boar spermatozoa are very susceptible to cryopreservation injuries and, for this reason, pig remains one of the few species in which fresh semen is still preferred to thawed one for routine artificial insemination (AI). The present work evaluated the effect of supplementing boar sperm thawing medium with Silvafeed SP (SSP), a mixture of Chestnut and Quebracho wood extracts (60/40 w/w) rich in polyphenols (92.4% tannin content) on in vitro fertilization (IVF) and on the following sperm parameters: sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function and lipid peroxidation (assessed by flow cytometry) and capacitation status (immunolocalization of tyrosine phosphorylated proteins). Thawed spermatozoa were incubated 1 h at 37°C in BTS without (CTR) or with (5, 10, 20 µg/mL) SSP. After incubation sperm suspension was divided in three aliquots: one was used for IVF trials, one for sperm analysis, and the last one was capacitated for 1 h at 39°C 5% CO2 in IVF medium. Sperm motility parameters, viability, acrosome integrity, mitochondrial functionality, lipid peroxidation and tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, were not influenced by SSP. However, oocytes inseminated with thawed spermatozoa pretreated with all the different SSP concentrations presented a significant (P < 0.01) increase in penetration rate compared to CTR. In addition, 5 µg/mL SSP exerted a positive effect (P<0.05) on the total efficiency of fertilization. These results encourage the use of SSP in the thawing medium since post-thawing fertility is a limit for the large-scale use of boar frozen semen.

Improvement of in vitro fertilization by a tannin rich vegetal extract addition to frozen thawed boar sperm

Boar spermatozoa are very susceptible to cryopreservation injuries and, for this reason, pig remains one of the few species in which fresh semen is still preferred to thawed one for routine artificial insemination (AI). The present work evaluated the effect of supplementing boar sperm thawing medium with Silvafeed SP (SSP), a mixture of Chestnut and Quebracho wood extracts (60/40 w/w) rich in polyphenols (92.4% tannin content) on in vitro fertilization (IVF) and on the following sperm parameters: sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function and lipid peroxidation (assessed by flow cytometry) and capacitation status (immunolocalization of tyrosine phosphorylated proteins). Thawed spermatozoa were incubated 1 h at 37°C in BTS without (CTR) or with (5, 10, 20 µg/mL) SSP. After incubation sperm suspension was divided in three aliquots: one was used for IVF trials, one for sperm analysis, and the last one was capacitated for 1 h at 39°C 5% CO2 in IVF medium. Sperm motility parameters, viability, acrosome integrity, mitochondrial functionality, lipid peroxidation and tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, were not influenced by SSP. However, oocytes inseminated with thawed spermatozoa pretreated with all the different SSP concentrations presented a significant (P < 0.01) increase in penetration rate compared to CTR. In addition, 5 µg/mL SSP exerted a positive effect (P<0.05) on the total efficiency of fertilization. These results encourage the use of SSP in the thawing medium since post-thawing fertility is a limit for the large-scale use of boar frozen semen.

Machine learning to further improve the decision which boar ejaculates to process into artificial insemination doses

Current artificial insemination (AI) laboratory practices assess semen quality of each boar ejaculate to decide which ones to process into AI doses. This decision is aided with two, world-wide used, motility parameters that come available through computer assisted semen analysis (CASA). This decision process, however, still results in AI doses with variable and sometimes suboptimal fertility outcomes (e.g., small litter size). The hypothesis was that the decision which ejaculates to process into AI doses can be improved by adding more data from CASA systems, and data from other sources, in combination with a data-driven model. Available data consisted of ejaculates that passed the initial decision, and thus, were processed into AI doses and used to inseminate sows. Data were divided into a training set (6793 records) and a validation set (1191 records) for model development, and an independent test set (1434 records) for performance assessment. Gradient Boosting Machine (GBM) models were developed to predict four fertility phenotypes of interest (gestation length, total number born, number born alive, and number of stillborn piglets). Each fertility phenotype was considered as a numeric and as a binary outcome parameter, totaling to eight different fertility phenotypes. Data used to further improve the decision process originated from four sources: 1) CASA information, 2) boar ejaculate information, 3) breeding value estimations, and 4) weather information. These data were used to create seven prediction sets, where each new set added parameters to the ones included in the previous set. The GBM models predicted fertility phenotypes with low correlations (for numeric phenotypes) and area under the curve values (for binary phenotypes) on the test data. Hence, results demonstrated that a combination of more data and GBM did not enable further improvement of the AI dose quality checks, resulting in the rejection of our hypothesis. However, our study revealed parameters affecting boar ejaculate fertility which were not used in today’s decision process. These parameters (listed in the top 10 in at least four GBM models) included one parameter associated with boar ejaculate information, two with breeding value estimations, five with CASA information, and one with weather information. These parameters, therefore, should be further investigated for their potential value when assessing the quality of boar ejaculates in daily routine AI doses processing.

Quality of frozen semen of Brahman bulls used for routine artificial insemination in Bangladesh

Background: Semen must be of good quality to achieve satisfactory conception rate in any artificial insemination (AI) programme. The objectives of the present study were to evaluate the quality of frozen semen of Brahman bulls and compare the same semen derived from different sources. Methods: Fifteen frozen semen straws of Brahman bulls derived from 3 different sources (5 straws from each source) were evaluated with respect to volume, motility, concentration of spermatozoa and morphology of spermatozoa. Volume of semen was determined by micropipette, concentration was evaluated by haemocytometer technique, motility and progressive motility were evaluated by computer assisted sperm analyser (CASA), spermatozoa with normal acrosome, midpiece and tail were evaluated in formol-saline fixed semen and spermatozoa with normal head morphology were evaluated by Farley staining technique. Moreover, presence or absence of bacteria in semen was evaluated by Gram’s staining technique. Results: The overall semen volume, concentration, motile percentage, progressive motile percentage, percentage of spermatozoa with normal acrosome, midpiece and tail, and percentage of normal head morphology were 0.19±0.01 ml, 30.87±3.4x106/ml, 56.47±24.2 (%), 16.23±9.8 (%), 88.23±2.1 (%) and 95.1±1.6 (%), respectively. When compared, similar volume of semen (0.19 ml) was found in all frozen semen derived from different sources. The difference in percentage of motile spermatozoa between source B and C was significant (P&lt;0.05). The difference in spermatozoa with normal acrosome, midpiece and tail in semen between source A and B was significant (P&lt;0.05). The difference in the percentage of progressive motile spermatozoa, the concentration of spermatozoa and normal head morphology in semen among different sources of semen was not significant (P&gt;0.05). The difference in percentage of motile spermatozoa, the percentage of progressive motile spermatozoa, the concentration of spermatozoa, normal acrosome, midpiece and tail, normal head morphology in semen between locally produced and foreign semen was not significant (P&gt;0.05). All examined 15 semen samples were negative for presence of bacteria. Conclusions: Although the quality of frozen semen derived from Brahman bulls seems to be within normal limit, the post-AI conception rate should be determined to evaluate the fertilizing capacity of the used semen.

Combined Single-Straw Packaging of Cryopreserved Stallion Epididymal Sperm and Separated Homologous Seminal Plasma

The aim of the present study was the investigation of stallion epididymal sperm frozen with seminal plasma within the same straw separated by an air bubble before freezing. We hypothesized that mixing both components after thawing improves the sperm quality compared with conventionally frozen-thawed epididymal sperm. Cryopreservation of epididymal sperm allows the preservation of spermatozoa in case of death or termination of the breeding carrier. However, these sperm lack contact with the seminal plasma, which plays an important role in the development of sperm functionality and the regulation of uterine inflammatory response after insemination. Motion characteristics, morphology, and viability were assessed in epididymal sperm mixed with seminal plasma after thawing (group SP) and compared with conventionally packed cryopreserved epididymal sperm that had no contact with seminal plasma (group FT). Total and progressive motility, curvilinear velocity (VCL), and average lateral head displacement were significantly higher in group SP (P < .05). During post-thaw incubation, VCL decreased in group FT but not in group SP (P < .05), and beat cross-frequency was significantly lower in group FT than in group SP after post-thaw incubation (P < .05). Straightness did not differ between the groups (P > .05). Although the percentage of tail abnormalities and isolated sperm heads increased significantly only in group SP after cryopreservation (P < .05), the proportion of morphologically abnormal and nonviable sperm was not affected by seminal plasma (P > .05). Mixing both components after thawing increased sperm motility and velocity but did not negatively affect morphology or viability. This packaging system is promising for routine artificial insemination of stallion epididymal sperm.

Biostimulation and reproductive performance of artificially inseminated rabbit does (Oryctolagus cuniculus)

Biostimulation is a non-hormonal and practical technique that has not yet been widely utilised when applied immediately before insemination to improve reproductive efficiency in livestock species. This study was conducted to determine the influence of short-term male biostimulation on behavioural and reproductive performance of inseminated rabbit does. A total of 142 female New Zealand White rabbits were randomly assigned to 3 groups. Females were either exposed to male odour (Odour group) or an adult aproned male (Male group), while the remaining does that were neither exposed to the male odour nor the adult male are considered the control group. All females were inseminated after the 2 h exposure session. Conception rates were determined by abdominal palpation 12 d after insemination. The results showed that conception rate of the male odour group (79.59%) was greater than that of male presence group (76.09%) and that of the control group (68.09%). Moreover, biostimulated does showed significant behavioural activities during the 2 h exposure session compared to the control group. Although no significant differences were recognised, litter size at birth and at weaning was slightly increased in biostimulated compared to control females. Nor were there any significant difference in serum oestradiol concentrations between treated groups. Conclusively, short-term 2 h biostimulation of rabbit does resulted in the appearance of various behavioural responses followed by differences in conception rates between groups after routine artificial insemination.

LOW TAURINE CONCENTRATIONS POSSITIVELY AFFECT RABBIT SPERMATOZOA PROPERTIES IN LATER TIME INTERVALS

The aim of this study was to determinate the effect of various taurine concentrations on rabbit spermatozoa in in vitro conditions with the potential use in routine artificial insemination. Taurine, amino acid distinguished by its strong antioxidant properties, has been widely tested on spermatozoa of all kinds of farm animals except for the rabbits. This study investigated differences in rabbit spermatozoa motility and viability between the control sample and samples enriched with taurine solutions: TAU3 (3.125 mM), TAU6 (6.250 mM) and TAU12 (12.5 mM). Ejaculates of nine adult rabbits were subjected to this study and observed at time intervals 0, 90, 180 and 270 minutes while cultivated at 39°C. For detection of viability the MTT test was used and motility parameters were carried out using CASA analysis. Experimental samples were compared against the control. Assessment of the mitochondrial toxicity revealed that the addition of taurine ensured the higher viability in supplemented samples however with no statistical significance. Monitored motility parameters (motility, progressive motility and velocity curved line of spermatozoa) showed that taurine treated spermatozoa have lower activity at the beginning of the study but along with increasing time the positive effect of taurine was induced. Significant (P

Postmating Endometritis and Pregnancy Rate Were Not Affected by the Addition to Frozen-Thawed Semen of Filtered Seminal Plasma When Mares Without Evidence of Endometritis Were Artificially Inseminated Once 40 Hours Post-Gonadotropin-Releasing Hormone Treatment

The main aim of this study was to evaluate the effect of the addition of seminal plasma (SP) to frozen-thawed semen on postmating endometritis (PME) and embryo recovery rate in mares when only one artificial insemination (AI) is performed. Forty hours following induction of ovulation, 15 fertile Standardbred mares were submitted to a single AI per cycle with frozen semen obtained from one of two different stallions, on two cycles, according to two different protocols: routine AI (200–280 × 106 frozen-thawed spermatozoa in 2 mL) and SP AI (200–280 × 106 of frozen-thawed spermatozoa in 2 mL to which 7.8 mL of frozen-thawed SP was added). Six and 20 hours after AI, mares were evaluated by ultrasound for the presence of uterine fluid. Six hours after AI, mares were also subjected to uterine lavage for the evaluation of the presence and number of inflammatory cells. Eight days after ovulation, pregnancy was diagnosed by embryo recovery. There was a significant effect of treatment on subjective motility, which was lower when SP was added (20%; interquartile range [IQR] 10) compared with undiluted semen (45%; IQR 10) (P < .05). There was no significant effect of stallion or treatment on PME or embryo recovery rate. In the mares and conditions of this study, the addition of SP to frozen-thawed semen had no effect on post-AI uterine inflammation and pregnancy rate.

Genome-wide mapping and estimation of inbreeding depression of semen quality traits in a cattle population

Inbreeding depression is known to affect quantitative traits such as male fertility and sperm quality, but the genetic basis for these associations is poorly understood. Most studies have been limited to examining how pedigree- or marker-derived genome-wide autozygosity is associated with quantitative phenotypes. In this study, we analyzed possible associations of genetic features of inbreeding depression with percentage of live spermatozoa and total number of spermatozoa in 19,720 ejaculates obtained from 554 Austrian Fleckvieh bulls during routine artificial insemination programs. Genome-wide inbreeding depression was estimated and genomic regions contributing to inbreeding depression were mapped. Inbreeding depression did affect total number of spermatozoa, and such depression was predicted by pedigree-based inbreeding levels and genome-wide inbreeding levels based on runs of homozygosity (ROH). Genome-wide inbreeding depression did not seem to affect percentage of live spermatozoa. A model incorporating genetic effects of the bull, environmental factors, and additive genetic and ROH status effects of individual single-nucleotide polymorphisms revealed genomic regions significantly associated with ROH status for total number of spermatozoa (4 regions) or percentage of live spermatozoa (5 regions). All but one region contains genes related to spermatogenesis and sperm morphology. These genomic regions contain genes affecting sperm morphogenesis and efficacy. The results highlight that next-generation sequencing may help explain some of the genetic factors contributing to inbreeding depression of sperm quality traits in Fleckvieh bulls.

Storage of sexed boar spermatozoa: Limits and perspectives

Despite the great potential application of sex-sorted spermatozoa in swine, the technology is not practiced in the pig industry because of technical factors and species-specific issues. The susceptibility of boar spermatozoa to stresses induced by the sorting procedure, the relative slowness of the sex-sorting process together with the high sperm numbers required for routine artificial insemination in pig are some of the main factors limiting the commercial application of this technology in pigs. This review briefly describes the damage to spermatozoa during sex sorting, focusing on an additional limiting factor: increased susceptibility of sexed boar spermatozoa to injuries induced by liquid storage and cryopreservation that, in turn, impairs sperm quality leading to unsatisfactory results in vivo. Strategies to extend the lifespan of sex-sorted boar spermatozoa and to improve their fertilizing ability after liquid storage or cryopreservation need to be implemented before this technology can be used in pig farms. In this regard, encapsulation in barium alginate membranes could be a promising technique to optimize the in vivo use of sexed boar spermatozoa, by protecting, targeting, and controlling the release of sperm into the female genital tract.

The effect of Curcuma longa extracted (curcumin) on the quality of cryopreserved boar semen.

The aim of this study was to determine the optimal concentration of curcumin needed for cryopreservation of boar semen. Semen samples (n = 9) were collected from nine Duroc boars which having proven fertility were used for routine artificial insemination. Semen samples were collected and divided into six groups (groups A-F) according to various concentrations of curcumin in freezing extender (i.e. 0, 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively). The semen was frozen by traditional liquid nitrogen vapor method and stored at -196°C in the liquid nitrogen tank. After storage, frozen semen samples were thawed at 50°C for 12 s and evaluated for progressive motility, viability and acrosome integrity. The present results indicated that the addition of curcumin at 0.25 (group C) or 0.50 mmol/L curcumin (group D) yielded the higher percentage of progressive motility (33.3 and 36.1%, respectively) (P < 0.001). A significantly higher percentage of acrosome integrity was found in groups B (29.7%), C (31.1%) and D (30.2%) than in the other groups (P < 0.01). However, there was no significant difference in percentage of viability among groups. In conclusion, addition to the freezing extender of curcumin during cryopreservation at a concentration of 0.25 or 0.50 mmol/L is the optimal concentration of curcumin for improving the quality (i.e. increased progressive motility and acrosome integrity) of cryopreserved boar semen.

Effects of acrosomal conditions of frozen-thawed spermatozoa on the results of artificial insemination in Japanese Black cattle.

The purposes of this study were to examine the relationship between male artificial insemination (AI) fertility and sperm acrosomal conditions assessed by new and conventional staining techniques and to identify possible reproductive dysfunctions causing low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions in Japanese Black bulls. We investigated individual differences among bulls in the results concerning (1) acrosomal conditions of frozen-thawed spermatozoa as assessed by not merely peanut agglutinin-lectin staining (a conventional staining technique) but also immunostaining of acrosomal tyrosine-phosphorylated proteins (a new staining technique), (2) routine AI using frozen-thawed spermatozoa as assessed by pregnancy diagnosis, (3) in vivo fertilization of frozen-thawed spermatozoa and early development of fertilized eggs as assessed by superovulation/AI-embryo collection tests and (4) in vitro fertilization of frozen-thawed spermatozoa with oocytes. The percentages of frozen-thawed spermatozoa with normal acrosomal conditions assessed by the abovementioned staining techniques were significantly correlated with the conception rates of routine AI, rates of transferable embryos in superovulation/AI-embryo collection tests and in vitro fertilization rates. These results are consistent with new suggestions that the distribution of acrosomal tyrosine-phosphorylated proteins as well as the acrosomal morphology of frozen-thawed spermatozoa are AI fertility-associated markers that are valid for the prediction of AI results and that low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions result from reproductive dysfunctions in the processes between sperm insemination into females and early embryo development, probably failed fertilization of frozen-thawed spermatozoa with oocytes.

Encapsulation of sex sorted boar semen: Sperm membrane status and oocyte penetration parameters

Although sorted semen is experimentally used for artificial, intrauterine, and intratubal insemination and in vitro fertilization, its commercial application in swine species is still far from a reality. This is because of the low sort rate and the large number of sperm required for routine artificial insemination in the pig, compared with other production animals, and the greater susceptibility of porcine spermatozoa to stress induced by the different sex sorting steps and the postsorting handling protocols. The encapsulation technology could overcome this limitation in vivo, protecting and allowing the slow release of low-dose sorted semen. The aim of this work was to evaluate the impact of the encapsulation process on viability, acrosome integrity, and on the in vitro fertilizing potential of sorted boar semen. Our results indicate that the encapsulation technique does not damage boar sorted semen; in fact, during a 72-hour storage, no differences were observed between liquid-stored sorted semen and encapsulated sorted semen in terms of plasma membrane (39.98 ± 14.38% vs. 44.32 ± 11.72%, respectively) and acrosome integrity (74.32 ± 12.17% vs. 66.07 ± 10.83%, respectively). Encapsulated sorted spermatozoa presented a lower penetration potential than nonencapsulated ones (47.02% vs. 24.57%, respectively, P < 0.0001), and a significant reduction of polyspermic fertilization (60.76% vs. 36.43%, respectively, polyspermic ova/total ova; P < 0.0001). However, no difference (P > 0.05) was observed in terms of total efficiency of fertilization expressed as normospermic oocytes/total oocytes (18.45% vs. 15.43% for sorted diluted and sorted encapsulated semen, respectively). The encapsulation could be an alternative method of storing of pig sex sorted spermatozoa and is potentially a promising technique in order to optimize the use of low dose of sexed spermatozoa in vivo.

Influence of seminal plasma on fertility of fresh and frozen-thawed stallion epididymal spermatozoa

The use of epididymal stallion spermatozoa for routine artificial insemination can secure easy future use of valuable genetics after unforeseen death or injury of a valuable stallion. The aims of this study were to (1) directly compare pregnancy rates for fresh and frozen-thawed stallion epididymal and ejaculated spermatozoa after conventional artificial insemination and (2) to investigate the effect of seminal plasma on the fertility of epididymal spermatozoa after insemination. Twenty-one mares were randomly assigned to three stallions. Mares were inseminated at five consecutive oestrous periods using fresh ejaculated spermatozoa (Fr-E, n = 18), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+, n = 12) or fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP−, n = 9), frozen-thawed ejaculated spermatozoa (Cr-E, n = 18), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+, n = 18) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP−, n = 15). Pregnancy examinations were performed 14 days after each ovulation. Pregnancy rates were 55.6% (Fr-E, 10/18), 75% (Fr-SP+, 9/12), 22.2% (Fr-SP−, 2/9), 38.9% (Cr-E, 7/18), 27.8% (Cr-SP+, 5/18) and 6.7% (Cr-SP−, 1/15). Overall pregnancy rates for fresh and frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma were significantly better than for epididymal spermatozoa that had never been exposed to seminal plasma ( P < 0.05). We conclude that the exposure of stallion epididymal spermatozoa to seminal plasma improves pregnancy rates.

Premature Capacitation of Frozen-Thawed Spermatozoa from Subfertile Japanese Black Cattle

Artificial insemination (AI) subfertility is an indication of failure of AI with frozen-thawed sperm classified as normal by conventional semen examination. Recently, 8 AI-subfertile Japanese Black cattle (S1-S8) were identified using the routine AI test or in vivo fertilization test, which included AI with frozen-thawed sperm of superovulated females and subsequent non-surgical recovery of presumptive zygotes. In the present study, we assessed capacitation states and in vitro oocyte penetration of frozen-thawed sperm from these bulls to estimate causal factors of AI subfertility. Frozen-thawed sperm from 8 AI-subfertile (S1-S8) and 9 fertile (F1-F9, control) bulls were washed and then used for a chlortetracycline (CTC) staining assay and in vitro fertilization test. The CTC staining assay revealed that approximately 50% of the sperm from 4 of the AI-subfertile bulls (S5-S8) were prematurely progressing into the capacitation state immediately after washing and resuspension in a CaCl(2)-lacking medium. In contrast, most of the sperm from the fertile bulls and other AI-subfertile bulls (S1-S4) remained uncapacitated. Addition of CaCl(2) to the medium effectively promoted a spontaneous acrosome reaction in the sperm samples from the AI-subfertile bulls (S5-S8). Moreover, the in vitro fertilization test showed that rates of sperm penetration into oocytes were significantly lower in sperm samples from the AI-subfertile bulls (S5-S8) than in the control sperm samples from the fertile bulls (F2-F4 and F7-F9). It has previously been suggested that prematurely capacitated sperm undergo a spontaneous acrosome reaction possibly due to uncontrolled influx of calcium ion, and consequently they possess relatively lower in vitro fertilizing ability. It is therefore possible that premature capacitation of sperm used for AI is a causal factor of subfertility of male Japanese Black cattle and a potentially good marker for identification of subfertile bulls for removal from AI programs.

Artificial insemination with frozen semen in dogs: A retrospective study of 10 years using a non-surgical approach

From 1994 to 2003, a total of 526 bitches of 99 different breeds were artificially inseminated in 685 estrus cycles with domestic ( n = 353) or imported ( n = 332) frozen-thawed semen from 368 males. The overall whelping rate was 73.1% and mean (±S.E.M.) litter size 5.7 ± 0.1 pups. The whelping rate was higher after intrauterine insemination (75.0%; n = 665) than after intravaginal insemination (10.0%, n = 20; P < 0.05). Insemination at the optimal time resulted in a higher whelping rate (78%, n = 559; P < 0.01) and larger litter size (5.8 ± 0.2; P < 0.05) than inseminations performed late or too late (55.7% and 4.5 ± 0.5, n = 61). Two inseminations ( n = 384) yielded a higher whelping rate ( P < 0.05) and mean litter size ( P < 0.01) than one insemination ( n = 241), 78.1% and 6.0 ± 0.2 and 70.5% and 5.1 ± 0.2, respectively. For inseminations performed at the optimal time, however, the whelping rate was not significantly different for bitches inseminated twice (79.3%, n = 358) versus once (76.8%, n = 168), but the litter size was larger (6.0 ± 0.2 and 5.3 ± 0.3). Semen classified as of poor quality (progressive motility <50% or percentage abnormal sperm >20%) resulted in a lower whelping rate ( P < 0.01) than semen classified as of good quality (progressive motility ≥50% and percentage abnormal sperm ≤20%), 61 and 77%, respectively. Small breeds ( n = 50) had a smaller litter size (3.9 ± 0.3; P < 0.01) than larger breeds (medium [5.7 ± 0.3, n = 94], large [5.9 ± 0.2, n = 295] or giant breeds [6.1 ± 0.5, n = 62] [ P < 0.01]). Bitches older than 6 years had a lower whelping rate (68.2%) than younger ones (77.0%; P < 0.05). The duration of pregnancy was longer ( P < 0.01) for bitches with a litter size of <3 pups (61.7 ± 0. 4 days, n = 30) than for bitches with larger litters (60.5 ± 0.1 days, n = 177). These results show the potential of transcervical intrauterine insemination for routine artificial insemination in dogs. The results with frozen semen inseminations were optimised by inseminating bitches ≤6 years old 2 and 3 days after ovulation with semen of good quality from males ≤8 years old.

Assessment of Some Morphofunctional Characteristics of Flow-Cytometrically Sorted and Stained Boar Spermatozoa

The possibility to predetermine the sex of offspring is one of the most ambitious targets of reproductive biotechnology. Besides the scientific implication in human medicine (sex-linked hereditary diseases), gender preselection of farm animals would have the most evident impact. For specific breeding purposes it may be convenient to have either males or females. Herd productivity can be increased by the selection of animals with the sex that fits the best with performance. Sex preselection could furthermore be successfully applied to the preservation of endangered species. The possibility to determine sex is primarily based on: (1) the use of PCR in blastomers taken from preimplantation embryos, characterized by technical and ethical problems, and (2) the separation of chromosome-X bearing spermatozoa from chromosome-Y bearing spermatozoa by cytofluorimetry on the basis of their different relative DNA content (Johnson and Pinkel, 1986; Johnson and Welch, 1999). The use of sexed spermatozoa represents in theory a simple procedure (Seidel and Garner, 2002); however, the real possibility of separating enough X-bearing spermatozoa from Y-bearing spermatozoa for routine artificial insemination in sow under field conditions is far from being a reality (Johnson, 2002; Rath et al., 2003). During the sexing procedure sperm cells undergo chemical, physical and electrical stresses that can induce sperm damage. The objective of this study was to determine the effect of the staining procedure, with or without flow cytometry, on boar sperm membrane integrity, mitochondrial activity and blastocyst development in vitro. The evaluation of these parameters was also performed on stained and sorted semen after 24 h of storage at 17◦C.