Number Of Accessory Spermatozoa Research Articles

Sire contribution to fertilization failure and early embryo survival in cattle

Despite passing routine laboratory tests of semen quality, bulls used in artificial insemination (AI) exhibit a significant range in field fertility. The objective of this study was to determine whether subfertility in AI bulls is due to issues of sperm transport to the site of fertilization, fertilization failure, or failure of early embryo or conceptus development. In experiment 1, Holstein-Friesian bulls (3 high fertility, HF, and 3 low fertility, LF) were selected from the national population of AI bulls based on adjusted fertility scores from a minimum of 500 inseminations (HF: +4.37% and LF: -12.7%; mean = 0%). Superovulated beef heifers were blocked based on estimated number of follicles at the time of AI and inseminated with semen from HF or LF bulls (n = 3-4 heifers per bull; total 19 heifers). Following slaughter 7 d later, the number of corpora lutea was counted and the uteri were flushed. Recovered structures (oocytes/embryos) were classified according to developmental stage and stained with 4',6-diamidino-2-phenylindole to assess number of cells and accessory sperm. Overall recovery rate (total structures recovered/total corpora lutea) was 52.6% and was not different between groups. Mean (± standard error of the mean) number of embryos recovered per recipient was 8.7 ± 5.2 and 9.4 ± 5.5 for HF and LF, respectively. Overall fertilization rate of recovered structures was not different between groups. However, more embryos were at advanced stages of development (all blastocyst stages combined), reflected in a greater mean embryo cell number on d 7 for HF versus LF bulls. Number of accessory sperm was greater for embryos derived from HF than for LF bulls. The aim of experiment 2 was to evaluate the effect of sire fertility on survival of bovine embryos to d 15. Day 7 blastocysts were produced in vitro using semen from the same HF (n = 3) and LF (n = 3) bulls and transferred in groups of 5-10 to synchronized heifers (n = 7 heifers per bull; total 42 heifers). Conceptus recovery rate on d 15 was higher in HF (59.4%,) versus LF (45.0%). Mean length of recovered conceptuses for HF bulls was not affected by fertility status. In conclusion, while differences in field fertility among AI sires used in this study were not reflected in fertilization rate, differences in embryo quality were apparent as early as d 7. These differences likely contributed to the higher proportion of conceptuses surviving to d 15 in HF bulls.

102 Sperm cryopreservation with a soy lecithin-based medium in black-footed cats (Felis nigripes) and sand cats (Felis margarita)

Felid semen has historically been frozen using an egg yolk-based cryopreservation medium (TEY). However, the use of egg introduces several potential concerns, such as variability in composition, microbial contamination, and regulatory issues. Our recent research has focused on developing an animal protein-free medium containing soy lecithin (SOY). Our studies revealed that SOY was superior to TEY for freezing domestic cat sperm and provided similar results for freezing ocelot, Pallas’ cat, and fishing cat sperm. The objective of this study was to compare SOY to the standard TEY for sperm cryopreservation in 2 wild cat species: the black-footed cat and sand cat. Semen was collected from adult male cats (n=6/species) via electroejaculation, split into 2 aliquots, centrifuged, resuspended in either SOY or TEY, slow-cooled, and frozen in straws over nitrogen vapor. Sperm motility [percent progressively motile (PPM); rate of progressive motility on 0-5 scale (RPM)] was evaluated at 0, 1, 3, 6, and 24h post-thaw and acrosome status (AC) was assessed at 0 and 6h post-thaw. Heterologous IVF was performed using oocytes collected laparoscopically from gonadotropin-treated domestic cats. At 48h post-insemination, Hoechst33342 staining was used to determine oocyte stage, number of blastomeres, and number of accessory sperm (AS) bound to the zona pellucida of embryos and mature oocytes. Percent progressively motile, RPM, and AC were analysed with repeated-measures ANOVA; embryo cleavage, blastomere number, and AS number were analysed with one-way ANOVA. All data are reported as least squares means±average standard error. In the black-footed cat, PPM, RPM, and AC of SOY-treated sperm (32.5±4.0% motile, 2.8±0.2 RPM, 41.8±4.1% intact; 0h) did not differ from TEY-treated sperm (44.2±4.0% motile, 2.8±0.2 RPM, 46.8±4.1% intact; 0h) at any post-thaw time point (P > 0.05). Similarly, in the sand cat, post-thaw PPM, RPM, and AC of SOY-treated sperm (36.7±5.2% motile, 2.6±0.2 progression, 53.3±5.8% intact; 0h) did not differ from TEY-treated sperm (45.8±5.2% motile, 2.8±0.2 RPM, 51.0±5.8% intact; 0h) at any time point (P > 0.05). In black-footed cats, neither embryo cleavage (34.1±10.9% SOY; 58.5±10.9% TEY), blastomere number (7.8±0.8 SOY; 6.3±0.8 TEY), nor AS (3.5±0.8 SOY; 1.7±0.8 TEY) differed between treatments (P > 0.05). Sand cat results were similar, with no difference between SOY and TEY for cleavage (44.7±10.8% SOY; 40.6±10.8% TEY) or blastomere number (7.4±2.0 SOY; 6.7±2.0 TEY) (P > 0.05), but AS was higher in SOY-treated sperm (4.3±0.2 SOY; 3.5±0.2 TEY, P=0.0183). These data collectively demonstrate that our SOY medium was an effective substitute to TEY for sperm cryopreservation in the black-footed cat and sand cat. The replacement of an egg yolk-based cryomedium with a chemically defined, animal protein-free alternative represents a significant advance in quality control and biosecurity for felid semen banking and should augment the use of assisted reproduction for population management of imperiled cats. Funded by the Institute of Museum and Library Services.

114 GETTING THE YOLK OUT: THE USE OF A SOY LECITHIN-BASED CRYOMEDIUM FOR SEMEN BANKING IN THE PALLAS’ CAT AND FISHING CAT

Classically, semen from nondomestic felids has been frozen in an egg yolk-based cryomedium (TEY). However, the inclusion of egg yolk is problematic, resulting in possible batch to batch variation, microbial contamination, and regulatory concerns with international semen transport. Our previous research revealed that a soy lecithin-based cryopreservation medium (SOY) was superior to TEY for freezing domestic cat sperm. However, there is limited information on the efficacy of SOY in nondomestic felids. In the present study, our aim was to assess the effect of SOY v. TEY on motility, acrosome status, and fertilizing capacity of frozen-thawed sperm in 2 wild cat species: the Pallas’ cat (Otocolobus manul) and fishing cat (Prionailurus viverrinus). Semen was collected via electroejaculation from male cats (n = 3/species), split into 2 aliquots, extended in either SOY or TEY (containing 4% glycerol), slow-cooled, and frozen in straws over liquid nitrogen vapor. Sperm motility (percent progressively motile, PM; rate of progressive motility on 1 to 5 scale, RPM) was evaluated at 0, 1, 3, 6, and 24 h post-thaw and acrosome status (AS) was assessed via fluorescein isothiocyanate-peanut agglutinin staining at 0 and 6 h post-thaw. Heterologous IVF was performed using oocytes (n = 10–15/experimental unit, 2 replicates) collected laparoscopically from gonadotropin-treated domestic cats. At 48 h post-insemination, Hoechst33342 staining was used to determine oocyte stage, number of blastomeres, and number of accessory sperm bound to the zona pellucidae of embryos and mature oocytes. The PM, RPM, and AS were analysed with repeated-measures ANCOVA, using pre-freeze values as a covariate. Embryo cleavage % and accessory sperm number were analysed with ANOVA. All data are reported as mean ± s.e. In the Pallas’ cat, PM, RPM, and AS of SOY-treated sperm (35.0 ± 7.6% motile, 2.8 ± 0.4 RPM, 41.2 ± 5.4% intact; 0 h) did not differ (P > 0.05) from TEY-treated sperm (36.7 ± 4.4% motile, 2.8 ± 0.4 RPM, 45.3 ± 9.4% intact; 0 h) at any post-thaw time point. Similarly in the fishing cat, post-thaw PM, RPM, and AS of SOY-treated sperm (38.3 ± 7.3% motile, 2.6 ± 0.2 progression, 20.0 ± 2.9% intact; 0 h) did not differ (P > 0.05) from TEY-treated sperm (31.7 ± 8.7% motile, 2.4 ± 0.3 RPM, 18.3 ± 6.4% intact; 0 h) at any time point. In the Pallas’ cat, neither embryo cleavage % (42.6 ± 7.0% SOY; 61.4 ± 14.8% TEY) nor accessory sperm number (12.2 ± 2.8 SOY; 18.1 ± 4.0 TEY) differed (P > 0.05) between treatments. Fishing cat results were similar, with no difference (P > 0.05) between SOY and TEY for cleavage % (60.4 ± 9.4% SOY; 47.9 ± 5.0% TEY) or accessory sperm number (4.6 ± 1.0 SOY; 5.5 ± 1.0 TEY). Collectively, these findings demonstrate that our SOY medium is an effective alternative to TEY for sperm cryopreservation in 2 nondomestic felid species. The replacement of an egg yolk-based cryomedium with a chemically defined, animal protein-free option represents a significant advance in quality control and biosecurity for cat semen banking and may enhance the use of assisted reproductive technologies for population management of imperiled felids. Research was funded by the Institute of Museum and Library Services.

Expression of estrus before fixed-time AI affects conception rates and factors that impact expression of estrus and the repeatability of expression of estrus in sequential breeding seasons

Expression of estrus after PG and before fixed-time AI has been reported to change the uterine environment, increase accessory sperm numbers, fertilization rates, and overall embryo survival. Thus, expression of estrus can strongly impact overall pregnancy success. Because of variation in percentage of beef females detected in estrus and number of animals per study, it can be difficult to detect a significant effect of estrus on pregnancy success. Thus, a meta-analysis was conducted using data from 10,116 beef females in 22 studies that utilized variations of the 5 most common fixed-time AI protocols (CO-Synch, 7-day CO-Synch+CIDR, 5-day CIDR, PG 6-day CIDR, and the 14-day CIDR protocols) to examine the effect of detection in standing estrus on subsequent fixed-time AI pregnancy success. A random-effects model was used to combine the studies/herds. The overall model indicated a positive effect of estrus on conception rates with cows detected in estrus before fixed-time AI having a 27% greater (P<0.05; 95% CI=22–32%) conception rate compared with those not detected in estrus. Next we determined factors that influenced expression of estrus. Data were available on 547 cows synchronized with a CIDR based fixed-time AI protocols and observed for estrus before AI during 2–4 breeding seasons. Analysis of these cows indicated that days postpartum (P=0.22) did not impact estrous expression. In contrast, BCS influenced estrous expression (P=0.04) with cows in a BCS of ≤4 (51±5%) having decreased expression of estrus compared to cows with a BCS>4 (≥70±4%). Initiation of estrous cycles before the breeding season also influenced estrous expression (P=0.03), with anestrous cows having greater expression of estrus compared with estrus-cycling cows (78±5% vs. 70±5%, respectively). In conclusion, among all currently recommended fixed-time AI protocols, cows detected in estrus before fixed-time AI had improved conception rates, with BCS and estrus-cycling status having the greatest influence on expression of estrus.

Effect of dietary organic zinc, manganese, copper, and cobalt supplementation on milk production, follicular growth, embryo quality, and tissue mineral concentrations in dairy cows

This study evaluated potential effects of organic trace mineral supplementation on reproductive measures in lactating dairy cows. Cows were blocked by breed and randomly assigned at dry-off to receive inorganic trace mineral supplementation (control; n = 32) or to have a portion of supplemental inorganic Zn, Cu, Mn, and Co replaced with an equivalent amount of the organic forms of these minerals (treatment; n = 31). Trace minerals were provided through control or treatment premixes fed at 100 g·cow(-1)·d(-1). Premixes were fed to dry cows (range = 40 to 72 d before calving) in 1.8 kg·cow(-1)·d(-1) concentrate pellets through a computer feeder to provide 40, 26, 70, and 100% of supplemented Zn, Mn, Cu, and Co, respectively, and to lactating cows (range = 69 to 116 d after calving) in a total mixed ration to provide 22, 14, 40, and 100% of supplemented Zn, Mn, Cu, and Co, respectively. Treatment increased milk production at wk 14 (P = 0.047) postcalving, milk urea N content (P = 0.039), and BW loss from calving to 1 mo postcalving (P = 0.040), and decreased milk fat percentage (P = 0.045) and BCS (P = 0.048). Treatment tended to increase milk production at wk 13 (P = 0.089) postcalving and endometrial tissue concentrations of Fe (P = 0.070), BW at mo 1 (P = 0.056), and milk protein percentage (P = 0.064). Treatment did not affect (P > 0.1) DMI, health events, first-wave follicular dynamics, first cycle luteal measures, embryo quality, liver trace mineral concentrations, or luteal trace mineral concentrations. Cows with a rectal temperature ≥39°C at the time of AI had a smaller percentage of fertilized entities (P < 0.001). However, of the entities that were fertilized, the percentage of viable embryos, embryo quality, accessory sperm number, and embryo cell number were not affected (P > 0.1) by treatment. We conclude that replacing a portion of inorganic supplemental trace minerals with an equivalent amount of these organic trace minerals (Zn, Mn, Cu, and Co) increased milk production in mid-lactation, but did not affect postpartum follicular dynamics, embryo quality, or liver and luteal trace mineral concentrations.

Period of dominance of the ovulatory follicle influences embryo quality in lactating dairy cows

Length of dominance of the ovulatory follicle and exposure to oestradiol (OE(2)) during proestrus can affect fertility. Lactating cows had their oestrous cycle pre-synchronized and were subjected to one of the four synchronization treatments. Cows in the oestrus detection (OD) treatment received GnRH on day 6 of the oestrous cycle, PGF(2alpha) 7 days later, and were inseminated at detected oestrus. The remaining cows were subjected to the Ovsynch (OVS) protocol (day 0 GnRH, day 7 PGF(2alpha), day 9 GnRH, and timed artificial insemination (AI) 12 h later) starting on day 3 (OVS3) or day 6 (OVS6 and OVS6E) of the oestrous cycle. Cows in the OVS6E treatment received an injection of 0.5 mg oestradiol cypionate 36 h before AI. Ovaries were examined by ultrasonography and blood was sampled for progesterone and OE(2) concentrations. Uteri were flushed 6 days after AI and recovered embryos-oocytes evaluated. Diameter of the ovulatory follicle at AI differed (P<0.01) among treatments, and it was the largest for OVS3 cows, which also had extended (P<0.01) length of follicular dominance. During proestrus, OD and OVS6E cows had increased (P<0.01) OE(2) concentrations. Fertilization was not altered by treatments, and maximum fertilization was achieved when the number of accessory spermatozoa was >7. Proportions of viable embryos in relation to embryos and embryos-oocytes recovered were smaller for OVS3 cows (P<0.01) than the other treatments, and embryos from OVS3 cows also had fewer (P<0.01) blastomeres and tended (P=0.09) to have a lower proportion of live blastomeres. Extending the period of follicle dominance did not alter fertilization but reduced (P<0.001) embryo quality. Embryo quality was compromised even when the dominance of the ovulatory follicle was extended by only 1.5 days.

Insemination factors related to timed AI in cattle

Six-day-old bovine ova/embryos were recovered non-surgically and used as biomonitors to evaluate time of artificial insemination. These embryos/ova provided information regarding fertilization status and embryo quality, as well as quantitative and qualitative data regarding associated accessory sperm. Both sperm access to the ovum (addressed by accessory sperm) and fertilization status/embryo quality were important in addressing pregnancy rate for specific intervals from the onset of estrus to insemination. Based on these biomonitors, early insemination failed to achieve optimum pregnancy rate due to inadequate access of sperm to the ovum (i.e., low fertilization rate, manifested by low accessory sperm numbers). However, embryo quality was high in early inseminations, which favors pregnancy. Late insemination failed to achieve optimum pregnancy rate (due to reduced embryo quality), however, sperm access to the ovum was highest. Thus, the selection of an insemination time to achieve optimum pregnancy rate appeared to be a compromise between the two extreme intervals. For timed-AI programs, consideration of the time of ovulation (and its variability) becomes important, in addition to conventional considerations, such as semen handling, site of insemination, and bull selection.

Effects of insemination–ovulation interval on fertilization rates and embryo characteristics in dairy cattle

The objective of this study was to examine effects of the interval between insemination and ovulation on fertilization and embryo characteristics (quality scored as good, fair, poor and degenerate; morphology; number of cell cycles and accessory sperm number) in dairy cattle. Time of ovulation was assessed by ultrasonography (every 4 h). Cows were artificially inseminated once between 36 h before ovulation and 12 h after ovulation. In total 122 oocytes/embryos were recovered 7 d after ovulation. Insemination–ovulation interval (12 h-intervals) affected fertilization and the percentages of good embryos. Fertilization rates were higher when AI was performed between 36–24 and 24–12 h before ovulation (85% and 82%) compared to AI after ovulation (56%). AI between 24 and 12 h before ovulation resulted in higher percentages of good embryos (68%) compared to AI after ovulation (6%). Insemination–ovulation interval had no effect on number of accessory sperm cells and number of cell cycles when corrected for embryo quality. This study showed that the insemination–ovulation interval with a high probability of fertilization is quite long (from 36 to 12 h before ovulation). However, the insemination–ovulation interval in which this fertilized oocyte has a high probability of developing into a good embryo is shorter (24–12 h before ovulation).

Incidence of Unilateral Fertilizations after Low Dose Deep Intrauterine Insemination in Spontaneously Ovulating Sows under Field Conditions

A new procedure for non-surgical deep intrauterine insemination (DUI) in unrestrained sows hormonally induced to ovulate, has been reported. In comparison with standard artificial insemination (AI), with this procedure, the sperm numbers inseminated can be reduced 20-fold without reducing the reproductive performance of these hormonally treated sows. The present study evaluated, using two experiments, the reproductive performance applying 20-fold different sperm numbers per AI dose using DUI or standard AI in spontaneously ovulating sows, under field conditions. In experiment 1, AI was applied to crossbred sows at 12, 24 and 36 h after onset of spontaneous oestrus using one of the following two regimes: (i) DUI (treatment) with 0.15 x 10(9) fresh boar spermatozoa in 5 ml of Beltsville thawing solution (BTS) extender (n = 95), and (ii) standard cervical AI (control) with 2.85 x 10(9) fresh spermatozoa in 95 ml of BTS extender (n = 95). The farrowing rates of the two groups of sows were statistically similar (NS). However, a decrease (p < 0.002) in litter size and the total number of pigs born alive was observed in sows inseminated with the DUI procedure. In experiment 2, 42 post-weaned oestrus sows were inseminated following the same design described for experiment 1 during spontaneous oestrus. On day 6 after onset of oestrus, the proximal segment of the uterine horns of the sows were flushed under surgery to retrieve eventual embryos and evaluate the success of fertilization per cornua (e.g. occurrence of effective uni- vs bilateral sperm transport rendering uni- or bilateral, complete or partial fertilization). Retrieved embryos were assessed for cleavage and number of accessory spermatozoa. Although identical overall pregnancy rates were achieved in both insemination groups, the percentage of sows with partial bilateral fertilization and unilateral fertilization was markedly higher (p < 0.05) in the DUI group (35%) compared with the control (standard AI) group (5%), with a consequent lower (p < 0.001) percentage of viable early embryos after DUI. The number of accessory spermatozoa in the zona pellucida of the embryos was highly variable, but higher (p < 0.001) in control animals than in DUI-AI. No accessory spermatozoa were found in oocytes retrieved from sows depicting unilateral fertilization. In conclusion, DUI in spontaneously ovulating sows with 0.15 x 10(9) spermatozoa renders similar farrowing rates but a lower litter size compared with use of standard AI with a 20-fold higher sperm dose. The lower litter size ought to be related to a decreased distribution of spermatozoa after DUI leading to a higher incidence of partial bilateral and unilateral fertilization.

10 TIMED ARTIFICIAL INSEMINATION IN CATTLE WITH REDUCED DOSAGE OF SPERMATOZOA

Estrus detection and determination of time of insemination are very important factors in reproduction management of cattle. Therefore an estrus synchronization schedule in combination with induction of ovulation and a single insemination at a predetermined time in dairy cattle was established to achieve high pregnancy rates (Kanitz etal. 2002 Reprod. Nutr. Dev. 42, 587–599; Becker et al. 2004). The aim of the recent study was to investigate the influence of the number of spermatozoa per insemination dosage on embryo development and the interrelationship between number of accessory sperms per embryo and its development using this schedule. In total 116 German Holstein heifers received GnRH (i.m.; 0.05 mg Gonavet®, Veyx, Schwarzenborn, Germany) 60 h after PGF2α application (i.m.; 0.5 mg Cloprostenol forte®, Jenapharm, Jena, Germany) administered between Days 8 and 14 of the estrous cycle. Artificial insemination was carried out 13 h after GnRH application. Three different dosages of spermatozoa (15 × 106, 5 × 106, and 1 × 106) from three ejaculates from four fertile bulls were used. Embryos and oocytes were flushed from the oviducts of animals ovulated (n = 106; ovulation rate 91.3%). Animals were slaughtered on Day 4 after insemination. The quality of the embryos and oocytes was evaluated by microscopic examination. Embryos were stained with Hoechst 33258 to verify the number of accessory sperm. The evaluation of the data was carried out with the GLM procedure of the statistics software package SAS® (SAS Institute, Inc., Cary, NC, USA). As a post-hoc test the Student’s t-test was used. Significance was set at P = 0.05. After flushing of 106 animals, 85 embryos and oocytes was recovered (recovery rate 80.2%). Relative to the three sperm concentrations, there were no significant differences among fertilization rates (92.3, 96.2, and 78.8%) and among the portions of normal developed embryos (84.6, 80.7, and 75.8%, all respectively) between groups. Interestingly, significant differences were found according to the mean number of accessory sperm/embryo (29.6 ± 8.4, 45.3 ± 8.6, and 6.5 ± 7.2, respectively) and in the portion of embryos without or with more than 10 accessory sperm/embryo. Results show that fixed-time insemination, independent of detection of onset of estrus can result in high fertilization rates. Insemination with dosages &lt;5 × 106 spermatozoa can reduce fertilization rates. Likewise, significant differences regarding fertilization rate were found after insemination of reduced sperm dosages of individual bulls. The number of accessory sperms/embryo seems to be an irrelevant parameter for quality of embryos produced under described conditions.

Accessory sperm: a biomonitor of boar sperm fertilization capacity

The number of accessory sperm found in the zona pellucida of porcine embryos was correlated to their individual quality and to the embryo quality range found within a single sow. Our goal was to determine whether accessory sperm counts provide semen evaluation with additional, useful information. Accessory sperm count was highest when only normal embryos were found in a given sow and diminished if oocytes or degenerated embryos were present ( P < 0.01). Within a given sow, normal embryos had higher ( P < 0.05) accessory sperm counts than degenerated embryos, although not when oocytes were also present. Fertilization capacity of sperm is optimal when only normal embryos are found in a given sow; this capacity is indicated by high accessory sperm counts. A decrease in fertilization capacity is reflected in diminishing accessory sperm counts. The boar had a significant effect ( P < 0.01) on accessory sperm count, but not on the percentage of normal embryos; this suggests that accessory sperm may be more sensitive indicators of the fertilization capacity of sperm than the percentage of normal embryos. We conclude that accessory sperm count can be used for the detection of compensable defects in sperm and is a valid parameter for assessing sperm fertilization capacity.

Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen–thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen–thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n = 28 and 31; year 2: n = 16 and 15, respectively) or laparoscopically inseminated (year 2: n = 13 and 14). In experiment 2, superovulated Belclare ( n = 4) and Suffolk ( n = 13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P < 0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P < 0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P < 0.02) but there was no difference after laparoscopic AI (62% versus 64%, P > 0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI ( P < 0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.

Assessing in vivo fertilizing capacity of liquid-preserved boar semen according to the 'Hanover gilt model'.

The goal of this study was to determine the ability of the Hanover gilt model to assess in vivo fertilizing capacity of preserved sperm and to consider whether any modifications to this model were needed. This model evaluates the fertilizing capacity of semen based on the fertilization rate, the rate of normal embryos and the accessory sperm count of 3-5-day embryos. Its distinguishing characteristics are the use of one-time insemination of sperm in reduced numbers, of spontaneously ovulating gilts and of ovulation detection through ultrasound examination of ovaries. Reduced sperm numbers allow for an accurate evaluation of the fertilizing potential of different semen treatments, thereby avoiding the compensatory effect of doses calibrated to maximize fertility. The model's usefulness was assessed in a trial run designed to compare the fertilizing capacity of liquid boar semen diluted into two different extenders. The diluent, the boar and the backflow, had no significant effect on any of the parameters studied. Gilts inseminated less than 24 h before ovulation had a significantly higher (p < 0.01) fertilization rate and accessory sperm cell count (p < 0.05) than those inseminated more than 24 h before ovulation. Very good/good embryos from homogeneous litters (only very good/good embryos were present) had a significantly higher (p < 0.01) accessory sperm count than those from heterogeneous litters (at least one embryo was of a different quality and/or oocytes were present). Both very good/good and degenerated/retarded embryos from heterogeneous litters had low accessory sperm numbers. This suggests that accessory sperm count is significantly related to the quality of the litter, but not to the quality of the embryo within gilts. It can be concluded that the Hanover gilt model is sensitive enough to show fertility differences (in this study, those associated with in vivo ageing of semen), while using relatively few gilts and little time.

Effect of boar exposure at time of insemination on factors influencing fertility in gilts.

The effect of boar exposure during artificial insemination (AI) on semen backflow, fertilization, and embryo quality was evaluated. Gilts (approximately 170 d) were induced into estrus with PG600, and ovulation was synchronized using hCG 72 h later. Estrus detection was initiated after PG600 and continued at 12-h intervals. At estrus, gilts were allotted to receive boar exposure (BE, n = 20) or no boar exposure (NBE, n = 20) during AI. Gilts receiving NBE were identified to be in estrus prior to AI and the boar was then removed for 1 h, whereas gilts in the BE group received 15 min of exposure during AI. Insemination occurred in crates at 12 and 24 h after onset of estrus with 3 x 10(9) sperm/80 mL. Backflow was collected continuously with samples taken at time 0, (during AI), and at 0.25, 0.5, 0.75, 1, 2, 4, and 8 h after first and second AI. The effect of treatment was evaluated for time of insemination (min), backflow (mL), and sperm in backflow samples. Oviducts were flushed 2 d after first AI to evaluate the effect oftreatment on fertilization rate, accessory sperm numbers on embryos (scored 1 to 5), and embryo quality. There was no effect of first or second AI; therefore, data were pooled. Average duration of AI was 3.7 +/- 0.2 min and was not influenced by BE (P < 0.10). However, during the initial stage of AI, BE reduced the volume of semen (18.6 vs 32.4 +/- 3 mL) and the number of sperm lost (0.8 vs 1.3 +/- 0.15 x 10(9) sperm) compared to NBE (P < 0.05). There was a treatment x time effect (P < 0.05) for volume of backflow. By 45 min, the BE gilts lost more volume (9.0 vs 3.6 mL) compared to the NBE group, but sperm loss did not differ. Between 1 and 8 h after AI, neither volume nor sperm loss was influenced by treatment. By 8 h, total leakage (65 vs 63 mL) and total sperm loss (1.6 x 10(9) vs 1.8 x 10(9) sperm) were not influenced by BE (P > 0.10). However, more accessory sperm (P < 0.01) were found on embryos for the NBE (> or = 11 sperm/embryo) compared to BE embryos (< or = 10 sperm/embryo). Despite this observation, percentages of fertilized embryos (99.5 +/- 0.5 %) and number of embryos (11.5 +/- 0.1) were not different (P > 0.10). In conclusion, AI in the presence of a mature boar did not affect total semen leakage, sperm loss, fertilized embryos, or embryo quality. The importance of boar exposure during insemination was evident from less leakage during insemination, but had no effect on fertility; this suggests that the elimination of boar exposure during AI may not be deleterious to reproductive performance.

Examination of the binding ability of bovine spermatozoa to the zona pellucida as an indicator of fertility.

Despite the development of many new techniques, laboratory assays still do not predict male fertility accurately. To identify targets for laboratory assessment, we first need to determine which steps in fertilization are most often defective in subfertile males. We developed a competitive in vitro fertilization assay in which spermatozoa from 2 different males, stained with different lipophilic dyes, are incubated together with oocytes in a droplet. By exposing mixed spermatozoa to the same oocytes, this assay controls for many of the variables of in vitro fertilization and should allow identification of the most common faulty steps in fertilization. The relationship of zona-binding ability to fertility is controversial. Therefore, as a first step, we determined if zona pellucida-binding ability, measured by this competitive assay, was related to bovine spermatozoal fertility. Fertility data were collected from 2 groups of bulls by 2 means of evaluation, nonreturn to estrus rates postinsemination and competitive insemination. In the nonreturn to estrus study, semen samples from 15 bulls were effectively ranked by zona-binding ability, using pairwise competitive in vitro zona-binding assays (R(2) = 0.84). However, this ranking was not significantly correlated with nonreturn rates (r = -0.04). In the competitive insemination study, semen samples from 8 bulls were effectively ranked by pairwise comparison using the competitive zona-binding assay (R(2) = 0.67). Again, this ranking was not significantly correlated to the competitive insemination index calculated for these bulls (r = 0.29). In the third study, we tested 3 bulls to determine if in vivo zona binding, assessed by the number of accessory spermatozoa, was correlated with in vitro zona binding. The number of accessory spermatozoa on oocytes recovered from cows after mating was not correlated with in vitro competitive binding of the spermatozoa. In conclusion, in vitro competitive zona binding was not correlated with bovine fertility or binding of accessory spermatozoa to oocytes in vivo.

Effect of Time of Insemination on Number of Accessory Sperm, Fertilization Rate, and Embryo Quality in Nonlactating Dairy Cattle

Two experiments were conducted to determine the effect of insemination time on number of accessory sperm per embryo (ovum), fertilization rate, and embryo quality. Semen was collected from three fertile Holstein bulls and cryopreserved in egg yolk-citrate-glycerol. In experiment 1, cows were continuously monitored for behavioral estrus by the HeatWatch estrous detection system and were artificially inseminated (AI) with one 0.5-ml straw (25 x 106 sperm) at the onset of estrus (AI 0h), 12h after onset (AI 12h), or received natural service at 0h (Nat 0h) from one of three bulls. From 150 inseminations, 115 embryos and ova (AI 0h: n = 39; AI 12h: n = 39; Nat 0h: n = 37) were recovered 6 or 7 d after insemination. Fertilization rates differed between treatments (AI 0h: 67%; AI 12h: 79%; Nat 0h: 98%). Median accessory sperm per embryo (ovum) also differed (AI 0h: 1; AI 12h: 10; and Nat 0h: 27) and paralleled the fertilization rate. Embryo quality was not affected by insemination time or natural service. In experiment 2, cows received AI at 0, 12, or 24h (AI 24h) after the onset of estrus as determined by HeatWatch. From 154 inseminations, 117 embryos and ova (AI 0h: n = 39; AI 12h: n = 39; AI 24h: n = 39) were recovered 6 or 7 d after insemination. Fertilization rates did not differ in experiment 2 (AI 0h: 66%; AI 12h: 74%; AI 24h: 82%); however, a trend toward a higher fertilization rate accompanied AI 24h. Median accessory sperm values increased from AI 0h (1) to AI 24h (4). Embryo quality declined with AI at increasing intervals after onset of estrus, as percentages of excellent and good, fair and poor, and degenerate embryos were as follows: 77, 15, 8; 52, 38, 10; and 47, 19, 34 for the 0-, 12-, and 24-h inseminations, respectively. Results indicate AI 12h after the onset of estrus provides a compromise between potential fertilization failure (AI 0h) and embryo failure (AI 24h), despite increased accessory sperm per embryo (ovum) after AI 24h. Artificial insemination 12h after onset of estrus should optimize fertility of dairy cattle through an acceptable fertilization rate, number of accessory sperm per embryo, and desirable embryo quality.

Towards the enhancement of pregnancy rate: The effect of insemination time on sperm transport, fertilization rate and embryo quality in dairy cattle

AbstractTo further identify factors which influence pregnancy rates, three experiments were conducted to determine the effect of insemination time on sperm transport, fertilization rate, and embryo quality. All cows were continuously monitored for behavioural oestrus by HeatWatch®, and received AI at heat onset (0 h after the first standing event), 12 h after onset, or received natural service at 0 hfrom one of three bulls (Exp. 1). In Exp. 2, cows received AI at 0 h, 12 h, or 24 h after the first standing event. On d 6 after insemination 115 embryos(ova) (Exp. 1) and 117 embryos(ova) (Exp. 2) were recovered from single-ovulating cows. For Exp. 1, median accessory sperm values were: 1 (0 h), 10 (12 h), 27 (natural service O h) (P &lt; 0.05). For Exp. 2, median accessory sperm values were: 1 (0 h), 2 (12 h), 4 (24 h) (P &lt; 0.05). Fertilization rates were: 67% (0 h), 79% (12 h), 98% (natural service O h) (P &lt; 0.05)(Exp. 1); and did not differ in Exp. 2. Embryo quality was not different in Exp. 1. In Exp. 2, percentages of excellent and good fair and poor, and degenerate embryos were: 77, 15, 8 (0 h), 52, 38, 10 (12 h), 47, 19, 34 (24 h) (P &lt; 0.05). In Exp. 3, 30 cows were superovulated and were inseminated once at either 0 h, 12 h, or 24 h after the first standing event. On d 6 after insemination, 529 embryos(ova) were recovered. Fertilization rates were: 29% (0 h); 60% (12 h); 81% (24 h)(P &lt; 0.01). Percentages of embryos with accessory sperm were: 5 (0 h); 8 (12 h); and 41(24 h) (P &lt; 0.01). Embryo quality was not affected by time of AI. We conclude that the time of insemination affects: 1) sperm transport as measured by median accessory sperm number (Exp. 1 and 2) and the percentage of embryos with accessory sperm (Exp. 3); 2) fertilization rate (Exp. 1 and 3); and embryo quality (Exp. 2).

Relationship of seminal traits and insemination time to fertilization rate and embryo quality

The nature of subfertility due to the male or inseminate is as complex as that of the female. Fertilization failure or failure in embryogenesis are both documented to be of seminal origin. Males also differ in the numbers of sperm required to reach their maximum fertilization rate. Males requiring more sperm would be considered to have compensable seminal deficiencies. These include a number of known (viability and morphology) and unknown factors (functional or molecular traits) precluding sperm access to the ovum or ability to engage the ovum sufficiently to initiate fertilization and the block to polyspermy. Differences in fertility among males or inseminates independent of sperm dosage are considered uncompensable. These deficiencies would be associated with fertilizing sperm that are incompetent to maintain the fertilization process or subsequent embryogenesis once initiated, with most failures occurring prior to maternal recognition of pregnancy. Such sperm would preempt fertilization by competent sperm. Chromatin aberrations in morphologically normal or near normal spermatozoa from abnormal semen samples appear to be the best candidates for the uncompensable deficiency. However, recognition of uncompensable or incompetent fertilizing sperm has not been achieved. Six-day-old non-surgically recovered bovine ova/embryos have been used to evaluate compensable and uncompensable seminal deficiencies as well as to test reproductive strategies. These ova/embryos provide information on fertilization status and embryo quality as well as quantitative and qualitative data regarding associated accessory sperm. Thus, they permit the separation of reproductive failure by fertilization from that by embryonic development. Accessory sperm number is positively associated with both fertilization rate and embryonic quality. Early insemination results in low fertilization rates (low accessory sperm number), but good embryo quality, whereas, late insemination results in high fertilization rates (high accessory sperm number), but poor embryo quality. Additional studies will be necessary to substantiate this model; however, if true, future research designed to improve results to artificial insemination should be tested by breeding early in estrus where sperm viability is most limiting and embryo quality is best.

Identification of embryo paternity using polymorphic DNA markers to assess fertilizing capacity of spermatozoa after heterospermic insemination in boars

Differences in sperm fertilizing capacity of males often remain undetected by routine semen parameters. Heterospermic insemination with equal numbers of spermatozoa from 2 males is an accurate method for assessing differences in fertility. Use of heterospermic insemination depends on a reliable, efficient assay to identify paternity of conceptuses or offspring. In this study, polymorphic DNA markers amplified by PCR were tested to determine paternity of Day 5 to 6 embryos. The fertilizing capacity of 2 boars (A and B) with similar semen parameters was compared after homospermic (n = 14 gilts) and heterospermic (n = 11 gilts) insemination. Single AIs were performed under suboptimal conditions using 1 × 10 9 spermatozoa at 12 to 24 h before ovulation to prompt differences in fertilization and to stimulate sperm competition. The fertilization rate and the number of accessory spermatozoa were determined in Day 5 to 6 embryos. Using 5 different polymorphic DNA markers, paternity could be determined in 95.8% of the embryos. Boar B sired significantly (P < 0.05) more offspring than Boar A after insemination with pooled semen, and this was reflected by a significantly (P < 0.05) higher number of accessory spermatozoa following homospermic insemination with semen from Boar B, although fertilization rates did not differ between the 2 boars after homospermic insemination. The results suggest that the viability of spermatozoa in the female reproductive tract contributes to differences in fertility rates of males with similar in vitro sperm quality parameters. The number of accessory spermatozoa is a more sensitive measure of boar fertility than the fertilization rate. Polymorphic DNA markers are suitable for verification of parentage even at a very early stage of embryonic development.

Effect of a deep uterine insemination on spermatozoal accessibility to the ovum in cattle: A competitive insemination study

A competitive insemination study was conducted to determine the effect of a deep uterine insemination on accessory sperm number per embryo in cattle. Cryopreserved semen of a fertile bull characterized by spermatozoa with a semi-flattened region of the anterior sperm head (marked bull) was matched with cryopreserved semen from an unmarked bull having spermatozoa with a conventional head shape. Using 0.25-mL French straws and a side delivery embryo transfer device, deep uterine insemination (0.125 mL deposited in each horn) was performed 2 cm from the uterotubal junction. Immediately after, the uterine body was artificially inseminated using semen (0.25 mL) from an alternate bull and a conventional insemination device. The complete dose (both inseminations) was 50 × 10 6 total sperm cells consisting of an equal number of spermatozoa from each bull. Single ovulating cows (n = 95) were inseminated at random with either the unmarked semen in the uterine body and marked semen in the uterine horn, or the unmarked semen in the uterine horn and marked semen in the uterine body. Sixty-one embryos(ova) were recovered nonsurgically 6 d post insemination, of which 40 were fertilized and contained accessory spermatozoa. The ratio and total number of accessory spermatozoa recovered was different among treatments: 62:38 (326) for the unmarked semen in the uterine body and marked semen in the uterine horn, and 72:28 (454) for the unmarked semen in the uterine horn and marked semen in the uterine body (P<0.05). Deep uterine insemination using this semen in a split dose and a side delivery device favors accessibility of spermatozoa to the ovum compared with conventional uterine body insemination.