The effect of fenceline bull exposure on expression of oestrus in dairy cows.

The objective of this study was to investigate whether dairy cows visit and interact with a fenceline-housed bull more during oestrus than outside oestrus and whether fenceline bull contact affects expression of oestrus. At one end of a free stall a fence with vertical open bars was placed behind which a bull could be housed, allowing interactions with the cows. A closed fence with two blinded entrances was placed before the fence, creating a contact area. The experiment consisted of three treatments; it started with the control treatment (no bull on the farm) and was followed by bull treatment (a bull housed behind the fence) and no bull treatment (a bull present on the farm but not housed behind the fence). Signs of oestrus were observed every 4h for 30min and cows were equipped with pedometers. On the day of oestrus, cows were more frequent in the contact area during the bull treatment (12.0+/-9.8 times) and the no bull treatment (13.9+/-10.2 times) than during the control treatment (2.6+/-2.5 times). The frequency of visits to the contact area was low and not different between treatments on the other days (2.2+/-1.9 times). More cows had direct contact with the bull on the day of oestrus (71.4%) compared to the days outside oestrus (21.4-30%). The duration of direct contact with the bull was highly variable between cows and did not differ between oestrus and non-oestrus days. Behaviour and duration of oestrus were not affected by treatment but the relative increase in number of steps during oestrus tended to be higher in the bull (5.5+/-0.2) and no bull treatment (5.3+/-0.3) than in the control treatment (4.6+/-0.3). In conclusion, dairy cows in oestrus seem to be attracted by a bull or by the expectation of the presence of a bull, but fenceline bull exposure does not affect behavioural expression of oestrus.

The acute effect of bull presence on plasma profiles of luteinizing hormone in postpartum, anoestrous dairy cows.

The objective of this study was to investigate whether bull exposure affects LH profiles in postpartum, anoestrous dairy cows. Eight cows between 10 and 17 days after parturition were used. On Day 1, blood samples were taken at 10 min intervals for 8 h. On Day 2, blood sampling continued at 10 min intervals and after 2 h a bull was introduced behind a fence, and blood sampling continued for another 8 h. Time of resumption of luteal activity was between 25 and more than 80 days after parturition for these animals and was not related (P>0.1) with frequency of LH pulses, amplitude of pulses and basal LH concentration on either Day 1 or Day 2. In 6 of the 8 cows, average and basal LH concentration were greater (P<0.001) during the 8 h of bull presence (0.56 +/- 0.33 and 0.39 +/- 0.26 ng/ml, respectively) compared to the 8 h without a bull (0.50 +/- 0.30 and 0.35 +/- 0.24 ng/ml, respectively). Pulse amplitude did not differ (P=0.85) between Day 2 (0.45 +/- 0.24 ng/ml) or Day 1 (0.45 +/- 0.14 ng/ml). LH pulse frequency was greater (P<0.1) on Day 2 (5.3 pulses/8h) compared to the Day 1 (4.6 pulses/8h). In conclusion, fenceline bull exposure early postpartum seems to have an acute effect on LH-release in anoestrous dairy cows. Whether sustained bull exposure can hasten first ovulation after calving through an effect on LH release in dairy cows is an interesting area of research.

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 4h). Cows were artificially inseminated once between 36h before ovulation and 12h after ovulation. In total 122 oocytes/embryos were recovered 7d after ovulation. Insemination-ovulation interval (12h-intervals) affected fertilization and the percentages of good embryos. Fertilization rates were higher when AI was performed between 36-24 and 24-12h before ovulation (85% and 82%) compared to AI after ovulation (56%). AI between 24 and 12h 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 12h before ovulation). However, the insemination-ovulation interval in which this fertilized oocyte has a high probability of developing into a good embryo is shorter (24-12h before ovulation).

Effects of exposure of epididymal boar spermatozoa to seminal plasma on the binding of zona pellucida proteins during in vitro capacitation.

The purpose of the investigation was to determine whether seminal plasma plays a role in the increase during in vitro capacitation of the number of boar spermatozoa with enhanced binding of zona pellucida proteins. Ejaculated spermatozoa and spermatozoa collected from the caudae epididymides of boars were incubated at 39 degrees C in a Tyrode's IVF medium. During incubation, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP), using a flow cytometer. Propidium iodide (PI) was included to simultaneously monitor cell viability. During incubation of ejaculated spermatozoa, a percentage of the spermatozoa expressed enhanced binding of FITC-sZP. The percentage of viable spermatozoa with enhanced binding reached a maximum of 37% (S.D.=8, averaged over five boars) after 2-3 h. In epididymal sperm, a similar maximum was observed after incubation in vitro, but a longer time of incubation was needed (6 h). Also, the rate of cell death of epididymal sperm was much lower than that of ejaculated sperm. When epididymal spermatozoa was exposed to seminal plasma in vitro, the time needed to reach a maximal percentage of viable spermatozoa with enhanced FITC-sZP binding was similar to that in ejaculated semen. However, the rate of cell death was still much lower than in ejaculated sperm. We concluded that the binding sites on the sperm surface that are involved in the increased binding of zona proteins during incubation under IVF conditions were not derived from the seminal plasma. The cellular processes leading to the increased binding capacity were accelerated by exposure of the sperm to seminal plasma.

Capacity of boar spermatozoa to bind zona pellucida proteins in vitro in relation to fertilization rates in vivo.

The purpose of this study was to determine variation among boars in the percentage of sperm in an ejaculate that express enhanced binding of zona pellucida proteins during treatment for capacitation in vitro, and to determine whether this relates to fertilizing ability in vivo. Ejaculates (n=35) were collected from 12 boars. A sample of each ejaculate was treated for capacitation in vitro. During incubation, the zona binding ability of spermatozoa was assessed at regular intervals with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP) and propidium iodide, using a flow cytometer. After incubation, a percentage of the sperm had enhanced FITC-sZP binding. The percentage of viable sperm with enhanced FITC-sZP binding, expressed as a percentage of the total sperm population, increased rapidly over the first 60 min and thereafter reached a plateau after 120-180 min. Averaged over all ejaculates, the percentage at 180 min was 46% (range 27-61%); this percentage was significantly different among boars. However, the variation between ejaculates within a boar was relatively small. There was no significant boar effect on the rate at which the percentage of viable cells with enhanced FITC-sZP binding reached the maximum. In ejaculates (n=14) from four boars (selected from the group of 12), we investigated the increase in the percentage of viable sperm with enhanced sZP binding during treatment for capacitation in vitro in relation to the ability to fertilize in vivo. Sows (n=44) were inseminated 4 h after ovulation with a suboptimal insemination dose (0.5x10(9) spermatozoa). Time of ovulation was determined using transrectal ultrasonography and sows were killed at 120 h after ovulation. The percentage of fertilized oocytes, embryo development, and numbers of accessory spermatozoa were determined. The percentage of spermatozoa that were viable and showed enhanced sZP binding after 180 min of incubation was 48 +/- 12% (range 28-56%). The percentage of fertilized oocytes was 85 +/- 27% and 64% of the sows had 100% fertilized oocytes. The percentage of sows with 100% fertilized oocytes correlated well (P< or =0.05, R2=0.98) with the percentage of viable spermatozoa with enhanced FITC-sZP binding after capacitation in vitro.

In vitro phagocytosis of boar spermatozoa by neutrophils from peripheral blood of sows.

A considerable number of spermatozoa are used in each sow in routine artificial insemination. However, within a few hours after insemination, many spermatozoa are phagocytosed by polymorphonuclear leucocytes. Some aspects of sperm transport in the female genital tract in the sow have been thoroughly investigated, whereas little is known about the mechanisms involved in the phagocytosis of spermatozoa, or about which spermatozoa (fresh, capacitated or dead) are the most susceptible to ingestion by polymorphonuclear leucocytes. In this study, phagocytosis was investigated by use of an in vitro phagocytosis assay. Polymorphonuclear leucocytes were challenged with either untreated, cold-shocked or frozen-thawed spermatozoa, or with spermatozoa that had been treated to induce capacitation in vitro. The influence of serum on phagocytosis was also investigated. Treatment of the semen to induce capacitation in vitro considerably reduced the phagocytosis of spermatozoa, whereas crude treatments like cold-shock or freezing and thawing reduced phagocytosis only in the first 15-30 min of incubation with polymorphonuclear leucocytes. Viable spermatozoa were phagocytosed mainly through a pathway that was independent of complement or other serum components (for example, antibodies). Complement had little effect on phagocytosis of spermatozoa, but did cause acrosomal exocytosis and cell death.

Enhanced binding of zona pellucida proteins to the acrosomal region of the intact boar spermatozoa in response to fertilizing conditions: a flow cytometric study.

In this investigation we sought to determine whether sperm capacitation in vitro is accompanied by changes in the functional presence of zona binding sites on the plasma membrane of boar spermatozoa. During sperm incubation at 39 degrees C in various modifications of a Tyrode's-based in vitro fertilization medium, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins, using a flow cytometer. Propidium iodide was routinely included to allow simultaneous assessment of membrane integrity; rhodamine-conjugated peanut agglutinin was used to assess acrosomal status. During incubation in the fertilization medium, a subpopulation of live acrosome-intact spermatozoa developed enhanced binding of the fluorescein-conjugated solubilized zona proteins. Microscopy revealed that the increase in cytometrically detected zona binding was paralleled by an increase in the area on the sperm head to which zona proteins bound, from the apical region to the whole of the acrosomal region. The changes were accelerated by phosphodiesterase inhibitors, were attenuated by omission of bicarbonate, and were completely inhibited by addition of EGTA. In the fertilization medium, numbers of sperm showing enhanced zona binding maximized after 60-90 min. This time course is somewhat similar to that reported by others for development of egg-penetrating ability in vitro. We suggest that the observed changes in zona binding ability bring about optimal sperm-egg attachment; they may also relate to induction of the acrosome reaction by zona pellucida components. In consequence, the zona binding changes may be an important part of the process by which the sperm acquires fertilizing ability as a result of capacitation.