Method agreement between three different chambers for comparative bull semen computer assisted sperm motility analysis.

The aim of this study was to compare computer assisted sperm analysis (CASA) results of frozen thawed bull semen using three different chambers. Sixty bull frozen semen samples were thawed (37°C; 30 sec), extended in PBS (30×106 spermatozoa/mL; 37°C) and incubated (37°C; 2 min). Each semen sample was analyzed by CASA [total motility, progressive (pro)/ non-progressive/rapid/medium/slow movement spermatozoa, VCL, VSL, VAP, ALH, BCF, LIN, STR, WOB and hyperactive spermatozoa] using three different chambers: a Makler® chamber (MC; 10 µm); a Leja 4 chamber slide (LC; 20 µm); and a Glass slide covered with a coverslip (GSC; 10.3 µm). The Makler chamber gave higher values compared to both the LC and GSC for almost all examined parameters. No systematic effect was evident between LC and GSC for VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF. Method agreement between MC and LC was generally moderate, between MC and GSC poor and between LC and GSC moderate to good. In general, narrower limits of agreement were found in samples with lower values. In conclusion, the CASA outcomes could be influenced by the analysis chambers. This finding should be taken into consideration when comparing results from different laboratories.

Selectivity of porcine zona pellucida to bind spermatozoa with normal chromatin structure.

This study aimed to investigate the selective ability of swine zona pellucida (ZP) to bind spermatozoa with normal nuclear chromatin. Ten ejaculates of four boars were used, while hemizona assay was applied for evaluation of binding capability. The results of this study showed that swine ZP has the ability to select spermatozoa with normal chromatin structure for sperm-zona binding process.

Relationship between sperm quality traits and field-fertility of porcine semen.

An investigation involving seven boars, active in artificial insemination, and 1,350 multiparous sows was conducted at a private farm and aimed at examining the relationship between sperm quality traits and boar fertility in terms of farrowing rate and litter size. This experiment was done for 6 months. The semen samples were evaluated for subjective sperm motility and concentration. Ejaculates with at least 1 x 10(8) sperm/mL and 70% sperm progressive motility were extended with a commercial medium to 30 x 10(6) sperm/mL and used for artificial insemination (AI). AI dose was 100 mL semen containing 3 x 10(9) spermatozoa. Aliquots of diluted semen were assessed for live morphologically normal spermatozoa (LMNS, eosin-nigrosin stain exclusion assay) and sperm chromatin instability (SCI, acridine orange assay). Farrowing rates according to different boar sperm varied (p < 0.001) from 59.3 to 88.92%. The mean values of LMNS (47.2 to approximately 76.5%) and SCI (0.16 to approximately 4.67%) differed significantly among boars. LMNS (r = 0.79, p < 0.05) and SCI (r = -0.90, p < 0.02) accounted for 62.2 and 81.7% of the variability in farrowing rates, respectively. After the combination of sperm traits, the relationship between percentage of LMNS with stable chromatin structure and farrowing rate was significant (r = 0.86, p < 0.05). The number of live piglets per parturition was not significantly correlated with sperm quality attributes. In conclusion, boar fertility after AI with freshly diluted semen can be predicted based on the evaluation of sperm morphology and chromatin integrity.

Effect of ram age on structural and functional competence of frozen-thawed spermatozoa in dairy sheep.

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen-thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1-2 years (young) or 4-5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of >or= 2.5 x 10(9) sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell medium and frozen in 0.25 ml straws. The end points of post-thawing semen evaluation were computer-assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per-cell analysis of lipid peroxidation using C11-BODIPY(581/591), sperm-hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen-synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non-capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = -0.63 to -0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.

Sperm chromatin stability during in vitro manipulation of beef bull semen.

Seven experiments were conducted to study the effect of freezing extenders, antioxidants, motility stimulants, thawing temperature, incubation temperature and time, centrifugation and capacitation on sperm chromatin instability (CI) as well as the influence of sperm CI on pregnancy rates of heifers (n = 360) after AI with frozen semen. Semen was collected once a week from Blonde d'Aquitaine and Limousine bulls (n = 3/breed) via an artificial vagina and only individual ejaculates (n = 300) of >0.3 x 10(9) sperm/ml and >or= 70% progressive motility were used. Sperm CI was evaluated by nuclear DNA susceptibility to acid-induced denaturation using acridine orange fluorescence and by chromatin susceptibility to decondensation using quantitative transmission electron microscopy. Bioxcell extender was better than AndroMed and egg yolk extenders in terms of low incidence of sperm CI in one bull (p < 0.05). Neither antioxidants (EDTA-2Na, Na-pyruvate and albumin) nor motility stimulants (caffeine and blood serum) had any significant effect on sperm CI. Thawing of frozen semen at 45 degrees C for 30 s decreased (p < 0.025) CI in one bull. Incubation of frozen sperm at 25 and 39 degrees C for 240 min increased sperm CI percentages from 3.47 +/- 0.48 and 4.50 +/- 0.41% to 6.70 +/- 0.36 and 9.71 +/- 0.53%, respectively (p < 0.001). Although centrifugation and removal of extracellular milieu increased CI of cooled sperm, it decreased CI of frozen-thawed sperm (p < 0.025). Follicular fluid as a capacitating agent destabilized chromatin structure (p < 0.001). Sperm vulnerability to CI had a negative impact (r(2) = 0.37-0.77, p < 0.001) on fertility of frozen ejaculates. In conclusion, in vitro manipulation of bovine semen can influence incidence of sperm CI, whereas integrity of sperm chromatin contributes significantly to heifers' fertility. We would recommend selection of the appropriate extender and thawing temperature for each bull together with careful manipulation of frozen semen to minimize damage of sperm chromatin.

Testing usability of butylated hydroxytoluene in conservation of goat semen.

The objective of this study was to investigate whether butylated hydroxytoluene (BHT) could be used as a suitable supporter or alternative of egg yolk during preservation of goat spermatozoa. Three in vitro experiments and a fertility test were conducted to evaluate the effect of BHT on viability of chilled-stored semen as well as motility and kidding rate of frozen-thawed spermatozoa. In the first two experiments, ejaculates (n = 30/experiment) were collected from 10 bucks, split, diluted with egg yolk-based and egg yolk-free extenders supplemented with or without 0.3, 0.6, 2, 5 and 8 mM BHT and stored at 5 degrees C for 168 h. In the third experiment, 30 ejaculates were collected from the above-mentioned bucks, split and diluted with egg yolk-free extenders supplemented with or without 0.3, 0.6 and 0.9 mM BHT and egg yolk-based extenders supplemented with or without 5 mM BHT. Diluted semen was cooled to 5 degrees C over a period of 4 h, frozen and thawed in the form of 0.3-ml pellets. In the fertility test, 75 ejaculates were collected from two proven fertile bucks, split, diluted with egg yolk-free extenders containing 0.6 mm BHT and egg yolk-based extenders supplemented with or without 5 mM BHT, frozen and thawed as described above. An insemination volume of 0.6 ml containing 120-140 x 10(6) progressively motile spermatozoa was used for a single cervical insemination of cloprostenol-synchronized does (n = 230). The results showed that addition of 5 mM BHT to egg yolk-deficient (2.5%) extenders significantly improved viability of chilled-stored semen together with motility (48.5%) and fertility (62.5%) of frozen-thawed spermatozoa. Replacement of egg yolk in semen extenders by 0.6 mm BHT could sustain not only viability of chilled-stored semen but also post-thaw motility (47.5%) and fertility (53.75%) of frozen-thawed spermatozoa. In conclusion, supplementation of semen diluents with BHT can ameliorate preservability of goat sperm.

Factors affecting chromatin stability of bovine spermatozoa.

The structural stability of transcriptionally inert paternal chromatin is of vital importance for the fertilization process and early embryonic development. Accordingly, a series of eight experiments were conducted during a 7-month period to investigate: (1) effects of bull breed, individuality, successive ejaculations, semen quality characteristics (SQC), semen dilution rates and hypothermic storage of semen in a Tris-egg yolk extender on incidence of sperm nuclear chromatin instability (NCI), and (2) effects of the interaction between variation of NCI within a frozen ejaculate and variation of oocytes quality due to maturation time and/or season on the efficiency of in vitro embryo production (IVEP). Semen samples were collected once a week from six bulls using an AV and only ejaculates (n=220) of >0.30x10(9) sperm/ml and >or=60% motility were used. NCI was measured by: (1) detection of lysine-rich histones in sperm chromatin using aniline blue staining, (2) sperm susceptibility to acid-induced nuclear DNA denaturation in situ using acridine orange test, and (3) sperm susceptibility to nuclear chromatin decondensation (NCD). Bovine oocytes (n=695) were matured in vitro for 18 or 24 h, fertilized after sperm selection through a swim-up procedure and cultured for 72 h. The results showed that the 2nd ejaculates were superior to the 1st ones with respect to chromatin stability. Dilution of semen to 49.67+/-8.56x10(6) sperm/ml (1:19) decreased resistance of sperm to NCD. Cooling of semen had no significant effect on chromatin stability. Cryopreservation of semen augmented sperm vulnerability to DNA denaturation. Improvement of SQC (semen volume, sperm motility, velocity, viability and morphological normalcy) was generally concomitant with increase of sperm resistance to NCI. While Blonde d'Aquitaine bulls had a resistance to NCD higher than Limousine bulls in fresh semen, the former showed a greater susceptibility to DNA denaturation than the latter in cooled semen. Individuality significantly influenced NCI. The variability of NCI within a frozen ejaculate affected efficiency of IVEP. Significant negative correlations were observed between incidence of NCI and both fertilization rate and developmental capacity of embryos after maturation of oocytes for 18 h. The significant variation in IVEP traits due to season was independent of the effect of sperm chromatin instability.

Study on the in vitro effect of zearalenone and alpha-zearalenol on boar sperm-zona pellucida interaction by hemizona assay application.

The mycotoxin zearalenone (zen) impairs fertility in farm animals. The aim of the present study was to investigate the effect of zearalenone and its major metabolite (alpha-zearalenol) on boar semen binding capacity, under in vitro conditions. Extended boar semen was exposed to three different concentrations of zen and alpha-zen (40, 60 and 80 microg ml(-1) of semen) for 1 h. Afterwards, the semen was washed and incubated with homologous oocyte hemizona for 4 h. A significant decrease (P < 0.001) in the number of tightly attached spermatozoa on the hemizona was obtained at concentrations of 60 microg ml(-1) and 80 microg ml(-1) of zen and alpha-zen. In conclusion, zen and alpha-zen affected the sperm-zona interaction by reducing the ability of boar spermatozoa to bind to the zona pellucida.

In vitro effect of zearalenone and alpha-zearalenol on boar sperm characteristics and acrosome reaction.

This study was conducted to determine the in vitro effects of three different concentrations (125, 187.5 and 250 microM in diluted semen) of zearalenone (zen) and alpha-zearalenol (alpha-zen) on boar sperm. Semen parameters such as motility, viability and spontaneous acrosome reaction were evaluated. From the results it was shown that both zen and alpha-zen affected the sperm characteristics significantly (p < 0.05), except for alpha-zen at the low concentration which did not decrease the percentage of live reacted spermatozoa significantly. In conclusion, zen and alpha-zen are directly toxic when they affect boar semen in vitro and consequently decrease the fertilization ability of the sperm. The higher the concentration of mycotoxin tested, the greater the decline of sperm parameters noticed. The influence of mycotoxins was found to be time- and dose-dependent.

Assessment of progesterone concentration using enzymeimmunoassay, for early pregnancy diagnosis in sheep and goats.

The objective of this study was to determine a value of serum progesterone (P4) concentration, assessed using an enzymeimmunoassay (EIA), for the early distinction between pregnant and non-pregnant ewes and goats. Adult, non-lactating ewes of Chios (n=53), Berrichon (n=30) and Sfakia (n=45) breeds were synchronized during the breeding season with progestagens and gonadotrophins and mated to fertile rams (Experiment I). Adult, lactating goats of Swiss breeds (Alpine and Saanen, n=104) and indigenous Greek breed (n=45) were synchronized during the transitional season with progestagens, PGF2alpha and gonadotrophins. Cervical artificial insemination (AI) with fresh semen was applied once, 42-44 h after sponge removal (Experiment II). Jugular blood samples were collected on day 19 after sponge removal (ewes) or on day 21 after AI (goats) and serum P4 concentration was determined by EIA. Progesterone concentrations >/=1.0, >/=1.5, >/=2.5 and >/=4.0 ng/ml were tested as indicative of pregnancy. Pregnancy diagnosis was verified on birth. In the case of sheep, using a discriminatory level of 2.5 ng/ml, overall accuracy of pregnancy diagnosis was 91.4% and predictive value of negative and positive diagnoses were 98.3 and 85.3%, respectively. In the case of goats, predictive value of negative diagnosis was 95.8 and 94.0% and predictive value of positive diagnosis 71.3 and 71.7%, for 1.5 and 2.5 ng/ml, respectively; overall accuracy was 79.2% using either level. The other discriminatory levels tested did not improve these results. A significant positive correlation was observed between P4 concentration and the number of lambs or kids born, and further analysis indicated that this relationship is not a simple linear function. Based on the results of this study, P4 concentrations of 2.5 ng/ml in the case of ewes and 1.5-2.5 ng/ml in the case of goats, determined with EIA, are proposed as discriminatory levels between pregnant and non-pregnant animals, at an interval of one oestrous cycle after service.

Progesterone concentration as an indicator of ovarian response to superovulation in Chios ewes.

We investigated the prediction of the ovarian response to superovulation using progesterone (P4) determination in Chios ewes. During the estrus period. estrus synchronization and multiple ovulations were induced in 100 non-pregnant, non-lactating Chios ewes by a combination of FGA-impregnated intravaginal sponges and 8.8 mg of ovine FSH. Laparoscopic insemination was conducted 24-28 h after the onset of estrus. A concentration of P4 was determined on Day 5 of the estrous cycle and on Day 6 the ovarian response was evaluated by counting the corpus lutea (CL); subsequently, embryo collection was performed. According to the response of their ovaries, ewes were allocated into four groups: A (n = 30); B (n = 37); C (n = 22); D (n = 11), with minimal (0-3 CL), moderate (4-8 CL), good (9-13 CL) or extreme (> 13 CL) ovarian response, respectively. In groups C and D, the mean blood serum P4 concentration (23.2 and 27.3 ng/ml, respectively) was higher (P < 0.001) than that in groups A and B (4.6 and 13.1 ng/ml, respectively); no difference was detected in blood P4 concentration between groups C and D. A strong linear relation (F < 0.00005) was found between blood P4 concentration and the number of CL, as well as between blood P4 and a dummy variable corresponding to poor (< 4 CL) or moderate/good/extreme ovarian response (>3 CL). Our results indicate that based on blood P4 measurement, it is feasible to identify ewes that should show the highest embryo recovery, while it is impossible to predict the exact number of CL formed.

Fertilization and embryo recovery rates in superovulated chios ewes after laparoscopic intrauterine insemination.

Forty superovulated dairy ewes of the Greek Chios breed were used in an experiment to evaluate the efficiency of laparoscopic intrauterine insemination on fertilization and embryo recovery rates as well as embryo quality. Estrus was synchronized by intravaginal progestagen impregnated sponges and superovulation was induced by administration of 8.8 mg o-FSH i.m. following a standard 8 dose protocol. A small volume (0.3 mL) of diluted fresh ram semen was deposited in each uterine horn 24 to 28 h after onset of the estrus by a laparoscopic technique. The animals were allocated randomly into two groups (Group A and B) of 20 animals each. In Group A, embryos were recovered 18 to 24 h after the intrauterine insemination and in Group B on Day 6. The average number of corpora lutea was 12.8 +/- 1.2 and 11.5 +/- 1.1 (+/- SEM); the overall embryo recovery was 66.4% and 57% and the percentage of recovered fertilized ova was 81% and 82.8% in Groups A and B, respectively. More fertilized ova were collected per ewe from Group A (P < or = 0.1). Results indicated that in Chios breed, superovulation using homologous FSH combined with laparoscopic AI leads to good ovarian response with satisfactory results in fertilization, embryo recovery and quality of embryos. This could lead to improved and more efficient methods for obtaining large numbers of high quality oocytes and embryos for embryo transfer programs which could contribute to genetic improvement and increase of the population size.

Effects of cadmium chloride administration on the macroscopic and microscopic characteristics of ejaculates from Chios ram-lambs.

The effects of cadmium chloride on the volume of the ejaculate, semen density, total number of spermatozoa per ejaculate, viability, grade of motility, and morphological abnormalities were studied in 3-month-old ram-lambs of the Chios breed. Two groups of seven animals each were used. For a period of 7 months, one group was treated with a daily oral dose (3 mg/kg b.w.) of cadmium chloride and the other group received the corresponding volume of doubly distilled water. Blood samples were collected for cadmium determinations, whereas semen was collected weekly. In the cadmium-treated animals, cadmium concentration in the whole blood was increased and the testes weight was lower. The volume of the ejaculate, the semen density and the total number of spermatozoa were significantly reduced by the administration of cadmium chloride. No differences were observed in the viability, the grade motility of spermatozoa, or the percentage of dead and morphologically abnormal spermatozoa between the control and the cadmium-treated animals. Histopathological examination in the cadmium-treated animals revealed the presence of lesions in the Sertoli cells, the seminiferous tubules, the primary and the secondary spermatocytes and the spermatides, whereas in the Leydig cells no significant lesions were evident.

Reproductive efficiency of three estrus synchronization schemes comprising fixed-time insemination in dairy cows.

The objective of this field trial was to compare the odds of pregnancy at 25 days (P25) and at 60 days (P60), of embryonic death (ED) between 25 and 60 days, and of non-pregnant cows returning to heat 20-25 days after artificial insemination (AI). Three fixed-time insemination protocols involving combined administration of either double gonadotrophin-releasing hormone (GnRH) and prostaglandins (A) or GnRH, human chorionic gonadotrophin (hCG) and prostaglandins (B), or double treatment with prostaglandins (C) were used. Cows included in the trial were selected from five herds, comprising a total of 735 (herd size range 85-250, median 120) Holstein dairy cows. Animals used for the study were multiparous, 4-7 years of age, had expressed at least one overt postpartum heat, were between 60 and 120 days of lactation and had at least one palpable corpus luteum and no gross morphological anomalies of their reproductive tracts. They were randomly allocated to receive treatment A (n = 89), B (n = 93), or C (n = 65). Pregnancy of cows not observed in heat 18-24 days post AI was diagnosed by transrectal ultrasonography at 25-30 days and confirmed by rectal palpation at 60-75 days post AI. Those that were detected in heat in the normal period post AI were re-inseminated. No differences were detected among the three treatment groups concerning the odds of P25 (P = 0.5), P60 (P = 0.2) and ED (P = 0.13). In contrast, non-pregnant cows that were administered treatment A were less likely to exhibit estrous signs in the normal period post AI than those administered treatments B (P = 0.02) or C (P = 0.005).

Ram seminal plasma and fertility: results from an ongoing field study.

The effects of partial replacement of ram semen diluent with ram seminal plasma on the fertility of ewes were studied. Crossbred Chios ewes (n = 152) were assigned to six groups. The oestrous cycles of the ewes were synchronised at the peak (Groups A, B, C and D) and at the end (Groups E and F) of the breeding season by means of intravaginal sponges impregnated with fluorogestone acetate (FGA) for 14 days. Four hundred IU of PMSG were injected intramuscularly at the time of sponge removal. Ewes of Groups A, C and E were artificially inseminated with ram semen diluted with skim milk extender, while those of Groups B, D and F with ram semen diluted with 50% skim milk and 50% ram seminal plasma. The addition of ram seminal plasma induced a significant increase (P < 0.05) in litter size in Groups B and D when compared with that of Groups A and C (1.85 and 1.88 vs. 1.39 and 1.52, respectively). This increase was not significant when insemination was performed at the end of the breeding season (2.0 vs. 1.4). These results indicate that the addition of seminal plasma can influence the fertility of ewes or the fertilising capacity of extended ram semen to some extent.