Agreement of different methods for assessment of progesterone concentrations in beef cattle serum.

Progesterone (P4) is responsible for the main reproduction processes. Concentration of P4 varies widely among different determination methods, and interpretation of these values may be difficult. The objective of the current study was to assess the agreement of three different enzyme immunoassays (ELISA) in relation to radioimmunoassay (RIA) of P4 concentration assessment of beef cow serum samples. Samples were collected randomly considering high (pregnant cows) and low (non-pregnant cows) P4 concentrations. Depending on the P4 assessment method, four groups were created as follows: Group 1 - direct samples assessed by ELISA, Group 2 - extracted samples assessed by ELISA, Group 3 - samples assessed by automated ELISA, and Group 4 - samples assessed by RIA. The mean progesterone concentration was 4.50 ng/mL, 1.24 ng/mL, 4.07 ng/mL and 4.39 ng/mL from Group 1 to Group 4, respectively. The mean difference (MD) between Group 1, Group 2 and Group 3 individually compared with Group 4 was -0.10 ± 1.24 ng/mL, 3.15 ± 3.58 ng/mL and 0.33 ± 1.42 ng/mL, and the 95% confidence interval (CI) for the differences (s) was from -0.99 to 0.78 ng/mL, from 0.59 to 5.71 ng/mL, and from -0.69 to 1.34 ng/mL, respectively. The confidence interval for the lower and upper limit of the agreement ranged from -4.12 to -1.05 ng/mL and from 0.84 to 3.91 ng/mL between Group 1 and Group 4, from -8.45 to 0.42 ng/ mL and from 5.88 to 14.75 ng/mL between Group 2 and Group 4, from -4.29 to -0.76 ng/mL, and from 1.41 to 4.94 ng/mL between Group 3 and Group 4. Our findings show that the best agreement with RIA was observed for Group 1 and Group 3, while the agreement in the extraction method was least accurate.

Effect of progesterone and oestradiol on sperm-zona binding and acrosome reaction in bovine spermatozoa after thawing.

This study was conducted assess spermatozoa binding capacity to the oocyte in the presence of 0.1 or 0.5 microg/ml progesterone (P4) or a combination of 0.5 microg/ml P4 with 0.1 microg/ml oestradiol (OE). The number of oocyte-bound spermatozoa in the presence of progesterone was significantly higher (p < 0.05 to p < 0.001) when compared with the control samples, that were incubated in the absence of P4. Spermatozoa binding to the zona pellucida (ZP) of the oocyte were concentration-dependent - significantly higher numbers of spermatozoa were bound in the presence of 0.5 microg/ml P4, when compared with that of 0.1 microg/ml P4. Oestradiol at 0.1 microg/ml concentration used impaired the effect of progesterone-mediated sperm-oocyte binding. The incidences of acrosome-reacted (AR) spermatozoa bound to the ZP - following 0, 60, 120 and 180 min. incubation in the presence and absence of 1 microg/ml progesterone was also assessed. Only at 180 min of incubation a higher (p < 0.001) incidence of the AR-spermatozoa was found in sperm-ZP complexes incubated in the presence or absence of progesterone, being 56.5 +/- 11.1 and 43.2 +/- 8.8 % respectively. In conclusion, progesterone enhances the sperm-ZP binding capacity. Progesterone affects the incidences of AR on zona-bound spermatozoa only after prolonged co-culture.

The effect of oestradiol, progesterone and heparin on bovine spermatozoa function after thawing.

The present experiment was designed to determine the effects of various biologically active substances, such as oestradiol (OE), progesterone (P4) and heparin (Hep) alone or in combination on sperm plasma membrane scrambling, capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa. Spermatozoa were incubated for 180 min in capacitation medium supplemented with (i) 1 mug/ml OE; (ii) 1 mug/ml P4; (iii) 1 mug/ml OE and 1 mug/ml P4; (iv) 1 mug/ml OE and 5 mug/ml Hep; (v) 1 mug/ml P4 and 5 mug/ml Hep; (vi) 1 mug/ml OE, 1 mug/ml P4 and 5 mug/ml Hep. At predetermined time intervals aliquots were taken to assess sperm plasma membrane scrambling, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment was aimed to study the effects of OE, P4 and OE/P4 as potential inducers of AR in Hep-capacitated spermatozoa. Plasma membrane scrambling was assessed by a flow cytometer, using Merocyanine staining. Acrosomal status and viability of spermatozoa were evaluated under epifluorescence microscope with Ethidium homodimer-1/peanut agglutinin fluorescein isothiocyanate staining method (EthD-1/PNA-FITC). The results show that OE, P4 and a combination of OE/P4 at concentrations used did not affect sperm viability. Heparin significantly (p < 0.001) increased sperm plasma membrane scrambling of OE and P4-treated spermatozoa. P4 significantly affected the rate of sperm capacitation (p < 0.001) and AR (p < 0.05), but OE expressed membrane-stabilizing properties (p < 0.05). It can be concluded that in frozen-thawed bovine spermatozoa OE presents plasma membrane stabilizing properties that can be abolished by Hep, but not by P4. Progesterone possesses capacitating and AR-inducing properties in frozen-thawed bovine spermatozoa that can be alleviated by OE.

Effect of exogenous progesterone on post-thaw capacitation and acrosome reaction of bovine spermatozoa.

The aim of this work was to study the effect of progesterone (P4) on capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa in vitro. Spermatozoa were incubated (0-180 min) in capacitation medium supplemented with 0, 0.1, 1.0 and 10.0 microg/ml of P4. At different time intervals aliquots were taken to determine sperm plasma membrane lipid destabilization, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment aimed to study the effects of P4, as potential inducer of AR in heparin-capacitated spermatozoa. The acrosomal status and viability of spermatozoa were evaluated under an epifluorescence microscope using Ethidium homodimer/peanut agglutinin fluorescein isothiocyanate staining method. Plasma membrane scrambling in spermatozoa was assessed by a flow cytometer, using merocyanine staining. The results show that P4 at the concentrations used had no negative effects on sperm viability. Progesterone significantly enhanced sperm capacitation (p < 0.001), but had no effect on plasma membrane lipid stability (p > 0.05) and did not significantly increase the AR of heparin-capacitated spermatozoa (p > 0.05). Progesterone displayed its effects in a dose-dependent manner with a maximum effect of 10 microg/ml P4 at 180 min of incubation. The results demonstrate that in cryopreserved bovine semen, P4 acts as capacitating, but not as an AR-inducing agent.

Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility.

This study investigated the use of annexin-V/PI assay to assess sub lethal changes in bull spermatozoa post-thawing, and to further relate these changes to results obtained by fluorometric assessment of sperm viability and sperm chromatin structure assay (SCSA), as well as field fertility (as 56-day non-return rates, 56-day NRR) after AI. Frozen-thawed semen samples were obtained from 18 Swedish Red and White bulls (one to three semen batches/bull) and fertility data was based on 6900 inseminations. The annexin-V/PI assay revealed that post-thaw semen samples contained on average 41.8+/-7.5% annexin-V-positive cells. Most of the annexin-V-positive cells were dying cells, i.e. also PI-positive. The incidence of annexin-V-positive cells was negatively related (r=-0.59, P<0.01) to the percentage of viable cells, as detected by fluorometry. The incidence of annexin-V-positive spermatozoa significantly correlated to the SCSA variable xalphat (r=0.53, P<0.05). The incidence of annexin-V-negative, dead cells was the only annexin-V/PI assay variable that correlated significantly with fertility both at batch (r=-0.40, P<0.05), and bull (r=-0.56, P<0.05) levels. Among sperm viability variables, subjectively assessed sperm motility (r=0.52-0.59, P<0.01), CASA-assessed sperm motility (r=0.43-0.61, P<0.05), and the incidence of live spermatozoa, expressed as total numbers (r=0.39-0.54, P<0.05), or percentage values (r=0.68-0.68, P<0.01), correlated significantly with field fertility both at batch, and bull levels. Among the SCSA variables, only the COMP alphat correlated significantly (r=0.33-0.51, P<0.05) with fertility results. The results indicate a certain proportion of bull spermatozoa express PS on their surface after thawing, e.g. they have altered membrane function, and that the incidence of such cells is inversely correlated to sperm viability, and positively correlated to abnormal sperm chromatin condensation since they eventually undergo necrosis.

Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls.

The present study examined the relationship between bull sperm characteristics post-thawing, after swim-up, and after challenge to calcium ionophore in relation to fertility (56-d nonreturn rates) after artificial insemination (AI). Spermatozoa from 25 semen batches derived from 15 Swedish Red and White AI bulls were evaluated with regard to post-thaw motility, membrane integrity, and migration through a swim-up procedure. The swim-up separated spermatozoa were assessed in terms of sperm concentration, viability and capacitation status as well as their response to exogenous calcium ionophore (A23187). Acrosome reactions were evaluated by fluorescence microscopy and flow cytometry. Sperm motility and viability post-thawing were significantly correlated with fertility. For the swim-up separated semen, significant correlations to nonreturn rates were found for concentration, viability, number of viable spermatozoa and sperm capacitation status (Pattern F and Pattern B). The only parameter significantly correlated to fertility after the ionophore challenge was the percentage of acrosome-reacted spermatozoa with remaining equatorial fluorescence, as assessed by fluorescence microscopy, but not by flow cytometry. The regression analysis showed that combining the results of sperm membrane integrity assessment post-thawing with those of capacitation status after swim-up provided the best prediction of fertility. The accuracy of prediction did not improve when these parameters were combined with the percentage of spermatozoa in which acrosome reaction was induced by ionophore challenge.

Relationship between the proportion of capacitated spermatozoa present in frozen-thawed bull semen and fertility with artificial insemination.

The objective of the present study was to confirm the presence of prematurely capacitated spermatozoa in frozen-thawed bull semen and to investigate the relationship of premature capacitation to the fertility of the respective semen. Twenty batches of frozen semen from young AI bulls of the Swedish Red and White breed with known fertility (expressed as 56-day non-return rates; 56 d-NRR) were tested using a Chlortetracycline (CTC) assay to assess capacitation status in frozen-thawed spermatozoa. The status of capacitation, as evidenced in this experiment, was further tested based on the hypothesis that capacitated spermatozoa present in frozen-thawed semen should undergo the acrosome reaction (AR) on co-incubation with homologous zona pellucida (ZP) glycoproteins. The percentage (mean +/- SEM) of uncapacitated, capacitated and acrosome-reacted spermatozoa in the frozen-thawed semen (n = 20) were 49.3 +/- 11.9, 36.3 +/- 8.3 and 14.2 +/- 11.9, respectively. On co-incubation with ZP, there was a significant increase (p = 0.001) in the proportion of spermatozoa undergoing the AR compared to the control with a concurrent decrease in the proportion of capacitated spermatozoa, suggesting that a proportion of capacitated spermatozoa were undergoing the AR. The proportion of viable, uncapacitated spermatozoa present in the frozen-thawed semen was correlated to the 56 d-NRR (n = 20, r = 0.5, p = 0.03). In conclusion, a proportion of spermatozoa in frozen-thawed semen was capacitated and the proportion of viable, uncapacitated spermatozoa present in semen was positively correlated to fertility.

Effect of cooling rates on post-thaw sperm motility, membrane integrity, capacitation status and fertility of dairy bull semen used for artificial insemination in Sweden.

We studied the effects of 2 different cooling rates during equilibration of semen from room temperature to 4 degrees C, at 4.2 degrees C/min (control split sample) or at 0.1 degree C/min (treatment split sample) on in vitro sperm viability post thawing and fertility after AI. Forty batches of split-frozen semen from 14 dairy bulls (Swedish Red and White breed) aged 14 to 16 m.o. or 66 to 79 m.o. were evaluated post-thawing for sperm motility (visual and computer-assisted sperm analysis [CASA], membrane integrity (fluorescent microscopy and flow cytometry post-loading with the combined fluorophores Calcein AM/EthD-1 and SYBR-14/PI); acrosomal status (with Pisum sativum agglutinin [PSA] staining); and capacitation status (CTC-assay). Fertility values (56-d nonreturn rate) of the slow cooling batches (treatment) were 0.4% units higher than for faster cooled (control) batches, but the difference was not statistically significant. Fertility values for the older bulls were 1.6% units higher than for the group of younger sires. No statistically significant correlations were found between semen viability parameters assessed in vitro and 56-d nonreturn rate. Visually assessed sperm motility, membrane integrity, capacitation and acrosomal status post-thawing did not differ significantly between cooling procedures, however the percentage of motile spermatozoa and the kinetic characteristics of spermatozoa--average path velocity (VAP), straight path velocity (VSL) and curvilinear velocity (VCL)--assessed by CASA differed significantly between cooling procedures. The results indicate that most of the in vitro sperm viability parameters post-thawing and the fertility results for bulls after AI did not differ significantly between the 2 semen cooling procedures tested.

Estimation of sperm viability in frozen-thawed semen from Swedish A.I. bulls.

From a total of 40 Swedish diary A.I. bulls, the number of viable spermatozoa in frozen-thawed semen was estimated using a commercial luminometer (SVT) to determine ATP contents and by fluorescence microscopic examination of samples stained with the combined fluorophore probes Calcein AM and Ethidium homodimer (CAM/EthD-1). The bulls were of two breeds (24 Swedish Red and White and 16 Holstein) and two age classes (1-2 and 6-10 years old, prior to and after progeny testing, respectively). The SVT-instrument was able to estimate a frequency of viable spermatozoa (47.0 +/- 14.4%; means +/- SD) close to that recorded by the fluorophore probes (spermatozoa with intact plasmalemma and progressively motile, 53.6 +/- 11.5%) and by subjective visual assessment of sperm motility (53.4 +/- 6.0%). The percentage of motile spermatozoa assessed under phase-contrast was positively (r = 0.33, P < 0.01) correlated with both the total ATP content, the percentage of viable spermatozoa recalculated from the SVT reading (r = 0.26, P < 0.05) as well as the frequency of spermatozoa having intact membranes as assessed by CAM/EthD-1 (r = 0.45, P < 0.001). The latter was also significantly correlated (r = 0.59, P < 0.001) with the total ATP contents. No significant variation in ATP contents was present between bulls, breeds or age. A significant difference, however, was seen between bulls for sperm membrane integrity. The results indicate that the SVT instrument seems to provide a quick and easy estimation of the number of viable spermatozoa in frozen-thawed bull semen. The assessment of membrane integrity with fluorophore probes allows the estimation of sperm subpopulations within the sample and seems therefore to be more useful and objective to determine sperm viability than visual assessment of sperm motility.

Influence of sperm number per straw on the post-thaw sperm viability and fertility of Swedish red and white A.I. bulls.

Semen from 5 Swedish Red and White bulls, approved in the fertility and progeny testing programme of a bull center, was split-frozen to produce straws with 15 or 10 x 10(6) spermatozoa each (control and treatment dose, respectively). Post-thaw sperm viability was evaluated by visual assessment of sperm motility (MOT), measurement of ATP (Adeinosin Tri Phosphate) contents by luminometry, assessment of membrane integrity with combined fluorophore probes [Calcein AM (CAM)/Ethidium homodimer (EthD-1)] and by using a hyposmotic swelling test (ORT). The straws were used for a total of 16,651 artificial inseminations (A.I.). No statistically significant difference was recorded between the 2 treatments for any of the post-thaw sperm viability parameters. In addition, a significant bull effect was evident for most post-thaw sperm traits assayed. Significant variation in overall fertility (56-days NRR) was recorded among the bulls used. A.I. with a reduced number of spermatozoa (10 x 10(6)/straw) resulted in a 2%-units decrease (n.s.) compared with controls (67.8% +/- 4.8%, means +/- SD) in overall fertility. In the control split-sample (15 x 10(6) spermatozoa/straw), MOT did not show any statistically significant correlation with fertility (r = 0.41, p = 0.07). However, MOT was correlated with the percentage of spermatozoa depicting progressive motility (category A1, r = 0.45, p < 0.05) as assessed with CAM/EthD-1. The latter was correlated with ATP contents (r = 0.57, p < 0.01), expressed as the percentage of viable spermatozoa. Both CAM/EthD-1 and ATP contents showed a statistically significant correlation with ORT (r = 0.45, p < 0.05 and r = 0.61, p < 0.05, respectively). In the straws with the reduced sperm number (10 x 10(6) spermatozoa), post-thaw motility was significantly correlated with fertility (r = 0.50, p < 0.05) and ATP-total contents (r = 0.48, p < 0.05). CAM/EthD-1 and ATP contents (as million viable spermatozoa) were significantly correlated (r = 0.47, p < 0.05), CAM/EthD-1 was significantly correlated with ORT (r = 0.48, p < 0.05). In conclusion, the results indicated that the freezing-thawing of straws with a reduced sperm concentration (10 x 10(6) spermatozoa) did not alter the post-thaw viability or overall fertility of the bull semen used. However, in view of the significant bull effect found in the limited population studied, we recommend that such a reduction in sperm number/straw be based on the fertility of the bull in question.