Fertility differences among male rabbits determined by heterospermic insemination of fluorochrome-labeled spermatozoa

Spermatozoa from different bucks were stained with different fluorochromes, mixed, and inseminated heterospermically. By altering the interval between insemination and luteinizing hormone injection, spermatozoa were allowed to reside in the female tract approximately 5, 10, or 15 h prior to ovulation. The number of functional spermatozoa, from each male of a pair used, that was transported to the site of fertilization was estimated by counting total number of differently stained spermatozoa that surrounded or fertilized each oocyte. Spermatozoa from split ejaculates within a male competed against each other equally, indicating that the staining procedure did not affect fertilization or functional spermatozoal transport rates. Three pairs of males with high initial semen quality (greater than 80% motility) differed in fertility primarily due to functional spermatozoal transport. Spermatozoal survival in the female tract and capacitation time played a role in differences in male fertility when heterospermic insemination occurred at variable times relative to ovulation. Differences in fertilization not accounted for by spermatozoal transport ratio raised the possibility that rate of egg penetration due to acrosomal enzyme differences may be important in determining male fertility. Therefore, total acrosin, hyaluronidase, and arylsulfatase activity in spermatozoa from specific bucks used in fertilization experiments were determined. Although there were trends favoring high fertility when enzyme content was higher, the difference was significant only for arylsulfatase in one buck.     

The usefulness of combining traditional sperm assessments with in vitro heterospermic insemination to identify bulls of low fertility as estimated in vivo

To date, no single in vitro assessment can estimate bull fertility. This research was aimed at evaluating the ability of a series of laboratory assessments to assign 50 Holstein Friesian bulls grouped as low (ER-NRR<-1.5), medium (-0.5相似文献   

Do sire-dam interactions contribute significantly to fertility comparisons in heterospermic insemination trials

The percentage of offspring sired after heterospermic insemination of equal numbers of spermatozoa is believed to be a very sensitive measure of relative in vivo fertility of the inseminated samples. The objective of these trials was to evaluate whether there was a detectable male-female interaction in the fertilizing ability of spermatozoa. If there was such an interaction, we reasoned that the paternity of offspring from individual females in a heterospermic trial the second year would be similar to the paternity of offspring in the same individual females the first year if the same ejaculates were used. Five groups of ewes were inseminated with different combinations of semen (a single Merino ejaculate from one of five rams randomly paired with five different pools of Suffolk semen) in a heterospermic trial. Those ewes conceiving the first year were inseminated in a second breeding season with the same combination of semen used previously. The percentage of lambs sired by each ejaculate/pool of ejaculates was calculated for all lambs born from all ewes inseminated with each semen combination. These percentages would be the expected ratios of Merino-sired:Suffolk-sired lambs if there is no male-female interaction. Ewes in each group were divided into two subgroups: those conceiving only Merino-sired lambs the first year and those conceiving at least one Suffolk-sired lamb the first year. The ratio of Merino-sired lambs:Suffolk-sired lambs did not differ in either subgroup from those expected if there was no male-female interaction. These results are consistent with the absence of a male-female interaction in relative fertilizing ability of spermatozoa.     

Study of fertilising capacity of spermatozoa after heterospermic insemination in rabbit using DNA markers

The aim of this study was to evaluate the fertilising capacity of males belonging to a rabbit line selected for growth rate using heterospermic insemination and genetic markers. Semen from five males was used to make pools of three of them, and to perform homospermic insemination. Insemination was carried out in receptive multiparous lactating does with 6 million spermatozoa per insemination dose. DNA from 360 young rabbits born from heterospermic insemination, 5 sires and 42 does were amplified to nine microsatellite loci for determination of the offspring rate per male. Although each female was inseminated with the same number of spermatozoa from each male (2 million from a total dose of 6 million), sperm from one male was always dominant, notable differences being observed in the offspring among the males with similar semen quality (83-68% from dominant male versus 31-0% from non-dominant, P<0.05 ).     

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 AI's were performed under suboptimal conditions using 1 x 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.     

Comparison of heterospermic and homospermic inseminations as measures of male fertility

This study attempted to determine a basis for the previously observed greater sensitivity of heterospermic tests when compared to homospermic tests for detecting differences in fertility between males. In theory, the results of heterospermic tests are an indication of the proportion of eggs fertilized per unit time whereas results of homospermic inseminations measure only the cumulative or final proportion of eggs fertilized. The fertilizing ability of sperm from males of CF1 and C57BL/6N strains of mice was compared homospermically using both relatively high and low concentrations of sperm and by measuring the proportion of eggs penetrated per unit of time. The fertilizing ability of sperm from these strains was also compared using heterospermic inseminations. When females were inseminated with a high concentration of sperm, males of both strains fertilized a high and indistinguishable percentage of eggs when examined after 30 hr. When females were inseminated with either a low concentration of sperm or when the proportion of eggs penetrated was measured at 5 hr, differences between strains of mice were distinguishable. Heterospermic insemination further magnified the observed difference between strains. The results of this study confirm that measuring the percentage of eggs fertilized per unit of time can enhance the magnitude of differences between males in fertility as compared to measuring only the final percentage of eggs fertilized. Measuring the percentage of eggs fertilized per unit of time does not, however, entirely account for the large differences observed between fertility of males when they are compared using heterospermic inseminations.     

The effect of exogenous testosterone on homospermic and heterospermic fertility in the cock

Cocks were administered testosterone (T) for 14 days via 0, 1, 2, or 4 Silastic capsules implanted subcutaneously. Each capsule released 1.04 mg of T per day. Concentrations of T in plasma and the proportion of eggs fertilized from homospermic insemination of hens were determined. Concentrations of T in plasma were variable and unaffected by treatment. The proportion of eggs that were fertilized by cocks decreased during treatment with 1 capsule, increased over the experiment in the group with 2 capsules, and increased after treatment ended in the group with 4 capsules. In heterospermic tests, cocks with distinguishable offspring were paired and semen was mixed within pairs. One cock in each pair received either 1 or 4 Silastic capsules containing T for 14 days; the other cock in the pair received none. The proportion of chicks sired by cocks treated with 1 capsule remained unchanged throughout the experiment, whereas the proportion sired by cocks treated with 4 capsules decreased markedly during the recovery period. The response to T was apparently dependent upon dosage and the sensitivity of the cock to T. The concentration of T in the plasma of the cock had little relationship to fertility. These results indicate that heterospermic insemination can be used as a sensitive method to detect the subtle effects of hormonal treatment.     

Artificial insemination of ewes with frozen-thawed semen at a synchronised oestrus. 2. Effect of dose of spermatozoa and site of intrauterine insemination on fertility

Three experiments were conducted to examine the effect of dose of inseminate, number of uterine horns inseminated and site of insemination on subsequent fertility of Merino ewes after synchronisation of oestrus, with progestagen-impregnated sponges (inserted for 12 days) and an injection of PMSG, and intrauterine insemination with frozen-thawed semen.The percentages of ewes lambing after insemination with 0.5, 5, 25 and 50 × 10⁶ spermatozoa were 29.3, 26.8, 56.3 and 62.1% respectively. A similar trend was observed in a second test resulting in 23.5, 38.8 and 53.1% ewes lambing after insemination with 5, 10 and 20 × 10⁶ spermatozoa respectively.The percentage of ewes lambing was higher for ewes inseminated in two uterine horns than one horn (76.8 vs. 44.9, P < 0.001). When semen was deposited in the tip, middle and bottom of the uterine horn, the percentages of ewes lambing and lambs born per ewe inseminated were 43.6 and 52.7, 52.8 and 84.9, and 41.2 and 64.7% respectively. Although site of insemination did not affect the percentage of ewes lambing, the percentage of lambs born per ewe inseminated was higher after insemination in the middle of the uterine horn than at the other sites (P < 0.001).     

Proportion of males sons-of-the-queen and sons-of-workers in Plebeia droryana (Hymenoptera,Apidae) estimated from data of an MDH isozymic polymorphic system

Stingless bees from 14 hives of Plebeia droryana were analysed for the MDH isozymic polymorphic system, which is controlled by four alleles, MDH1-1, MDH1-2, MDH1-3 and MDH1-4. The hives came from four different localities in Brazil and at least 15 drones were tested from each one, to estimate the proportion of them that are sons of the queen or of workers; the obtained values were 83.8% (range 100% to 43%) and 16.2% (range 0% to 57%), respectively. It is suggested that male-producing workers evolved from the need to preserve xo-heteroalleles.     

Correlations between heterospermic fertility and assays of porcine semen quality before and after cryopreservation

The relative fertilizing potential of frozen-thawed semen from four black and four white boars was determined following heterospermic insemination. A heterospermic index (HI) was computed for each of the 16 possible pairs of black and white boars. Correlation coefficients were computed between the HI and several in vitro tests of semen quality before and afttr cryopreservation of the semen. For the in vitro tests before cryopreservation, the HI were negatively correlated (-0.57) with spermatozoal motility before cooling the semen, but they were not correlated with spermatozoal motility after cooling to 5 degrees C. After freezing and thawing, the HI were correlated with the following in vitro tests: spermatozoal motility (0.50), spermatozoa with either normal or damaged apical ridges (0.31), spermatozoa with missing apical ridges (-0.51), spermatozoa filtered through sephadex columns (0.32), spermatozoa with acrosin-activity (0.38), percentage of maximal releasable glutamic oxalacetic transaminase (GOT) present extracellularly (0.54), spermatozoal intracellular GOT (-0.57), spermatozoa bound per zona-free hamster oocyte (0.64), and percentage of zona-free hamster oocytes penetrated (0.75). The HI were not correlated with the following in vitro tests after freezing and thawing: spermatozoa with normal apical ridges, damaged apical ridges and loose acrosomal caps, extracellular and maximal releasable GOT, and the number of penetrations per zona-free hamster oocyte. The multiple regression correlation coefficient between the HI and four selected variables from three in vitro tests was 0.94. This high correlation indicated that the fertilizing potential of the semen could be accurately predicted with four variables that appeared to measure different properties of the spermatozoa.     

Post-thaw survival of ram spermatozoa and fertility after insemination as affected by prefreezing sperm concentration and extender composition

A study was conducted to investigate the effects of prefreezing sperm concentration using two extenders on post-thaw survival and acrosomal status of ram spermatozoa (Experiment 1) and fertility after intrauterine insemination with differing doses of semen (Experiment 2). In autumn (Northern hemisphere), semen was collected by artificial vagina from 8 adult Leccese rams and ejaculates of good quality semen were pooled. Two extender systems for cryopreservation were considered, one based on milk-lactose egg yolk (Milk-LY) and the other based on tris-fructose egg yolk (Tris-FY). Experiment 1 (2 x 6 factorial scheme) examined the in vitro characteristics of spermatozoa in relation to the Milk-LY and Tris-FY extenders and six prefreezing sperm concentrations (50, 100, 200, 400, 500 and 800 x 10(6) spermatozoa/mL). Experiment 2 (2 x 4 factorial) evaluated the influence of the Milk-LY vs Tris-FY extenders and four doses (20, 40, 80 and 160 x 10(6) spermatozoa/0.25 mL) corresponding to prefreezing spermatozoa concentrations of 100, 200, 400 and 800 x 10(6) spermatozoa/mL, on fertility of ewes inseminated in uterus by laparoscope. Prefreezing sperm concentration influenced (P < 0.01) freezability of spermatozoa and affected negatively all the in vitro parameters at 800 x 10(6) spermatozoa/mL. Overall, Milk-LY tended to ensure higher viability and acrosomal integrity of spermatozoa after thawing at the intermediate sperm densities (range 100 to 500 x 10(6) spermatozoa/mL). At 500 x 10(6) spermatozoa/mL concentration corresponded the best condition for survival of spermatozoa (71.2%), acrosome integrity (71.5%) and acrosomal loss (6.0%). At the lowest sperm concentration (50 x 10(6) spermatozoa/mL), Tris-FY resulted in a higher survival rate than Milk-LY (61.3%, P < 0.05) and lower acrosomal loss (9.7%, P < 0.05). Milk-LY supported spermatozoa motility better than Tris-FY after incubation at sperm concentration between 50 and 400 x 10(6) spermatozoa/mL (0.05 > P < 0.01). Semen doses of 20 to 40 x 10(6) spermatozoa/ewe provided satisfactory fertility rates (64 to 81%). The increase of inseminate doses to 160 x 10(6) spermatozoa/ewe failed to improve fertility, actually tending to decrease lambing rates.     

Morphology of spermatozoa in semen from stallions of normal fertility.

Semen samples were collected from 3 fertile stallions by means of an 'open' artificial vagina and examined under scanning and transmission electron microscopy. The stallion spermatozoon has many features in common with that of other mammals but differs specifically in that it has an asymmetric head, an abaxial position of the tail and an acrosome of small volume. The presence of microtubules in the neck is also a characteristic of stallion spermatozoa.     

Platelet-activating factor content in boar spermatozoa correlates with fertility

The objective of this study was to evaluate the level of platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] content in spermatozoa between two groups of boars that differ in farrow rate percentages. The boar farrow rate was defined as High if it was > or = 70% and Low if it was < 70%. Fresh, extended semen was collected from sexually mature boars and used in the PAF extractions. Platelet-activating factor was detected in all semen samples assayed. The amount of PAF detected in spermatozoa obtained from the High group ranged from 1.90 to 11.30 pM/10(6) cells. The level of PAF in the Low group ranged from 0.92 to 4.96 pM/10(6) cells. Regression analysis revealed a positive (R2 = 0.369) and significant (P = 0.021) relationship between PAF content in boar spermatozoa and farrow rate. Spermatozoa-derived PAF levels (mean +/- SEM) were significantly higher (P = 0.015) in the High-farrow group (6.75 +/- 1.25 pM/10(6) cells) than in the Low-farrow group (2.45 +/- 0.51 pM/10(6) cells). The PAF content in spermatozoa was significantly higher (P = 0.035) in the High-average (> or = 10.5/litter) number of piglets born group (5.78 +/- 1.24 pM/10(6) cells) than in the Low-average (< 10.5/litter) number of piglets born group (3.34 +/- 1.19 pM/10(6) cells). Additionally, PAF content in spermatozoa was significantly higher (P = 0.034) in the High-average (> or = 9/litter) number of piglets born alive group (6.82 +/- 1.35 pM/10(6) cells) than the Low-average (< 9/litter) number of piglets born alive group (3.00 +/- 0.87 pM/10(6) cells). The data demonstrate that PAF is present in boar spermatozoa and that levels are significantly higher in individuals with a high-farrow rate status and high-number of piglets born and born-alive.     

In vitro fertilization as a predictor of fertility from cervical insemination of sheep

The objective of this study was to determine if the quality of frozen-thawed ram semen could be effectively evaluated through in vitro fertilization (IVF) procedures prior to insemination as a means of improving pregnancy rate. In experiment 1, frozen semen from four Belclare rams was assessed using IVF and was used for cervical insemination of ewes (n = 181) in 13 pedigree Belclare flocks. There was a significant association between IVF score (proportion of oocytes cleaved at 48 h post insemination) and non-return rate (P < 0.001). For experiment 2, semen from nine Belclare rams was evaluated by IVF and semen from rams with the highest (n = 3) and lowest (n = 2) IVF scores was used for cervical insemination of ewes (n = 111) under experimental conditions. Differences in pregnancy rates between individual rams did not reach significance. Experiment 3 was designed to determine if differences detected between rams at field level could be accurately identified via IVF evaluation and involved frozen semen from eight Norwegian rams of known field fertility (non-return rates ranged from 45.7 to 73.8%). IVF score did not reflect the differences in field fertility. In the final experiment six of the eight Norwegian rams involved in experiment 3 were selected based on IVF score (three highest and three lowest) and their semen was used for cervical insemination (n = 90 ewes). While significant differences in pregnancy rate were found between individual rams (P < 0.02, range: 12.9-65.8%) they were not associated with IVF score. Ewe breed had a significant effect (P < 0.003) on pregnancy rate in both experiments 2 and 4. In conclusion, there was no evidence from this study that the evaluation of semen quality through IVF provided a useful predictor of pregnancy rate under field conditions. It may be that the IVF procedures as used routinely, which are essentially designed to maximize blastocyst yields rather than for detecting differences in fertilizing ability between batches of sperm, need to be modified.     

Transuterine transport of spermatozoa after artificial insemination in heifers

Eight heifers were artificially inseminated with frozen-thawed semen during heat. Semen was deposited in one of the uterine horns. The animals were slaughtered 2 h after insemination and the genital tract was flushed. Sperm concentration in the flushing fluid was estimated by haemocytometric counting.There was a considerable transport of spermatozoa from the site of semen deposition to the uterine horn and oviduct on the opposite side. Spermatozoa were recovered from all parts of the oviduct (infundibulum, ampulla and isthmus) and distal and proximal parts of the horn on the non-inseminated side. In 7 out of 8 heifers more spermatozoa were recovered from the side of the tract opposite to insemination than from the inseminated side, although the differences were small in 2 animals. No clear relationship could be seen between ovarian activity and distribution of spermatozoa.