Contents Some stallions produce ejaculates of low quality and/or low fertility when used for artificial insemination (AI). The purpose of these five case studies was to use Single Layer Centrifugation (SLC) to select the best spermatozoa from ‘problem’ ejaculates for subsequent use in AI. Sperm quality, in terms of motility, morphology and chromatin integrity, was improved in the SLC‐selected samples compared to the corresponding uncentrifuged samples, with the exception of one stallion thought to have ampullary stasis. In this stallion, neither the incidence of spermatozoa with detached heads nor the proportion of damaged chromatin was decreased by SLC, in contrast to previous results. Pregnancies were obtained after using SLC‐selected spermatozoa from the five stallions for AI, indicating that the spermatozoa were functional after SLC. Overall, the results suggest that SLC may be useful when preparing AI doses from some ‘problem’ ejaculates.
Previously, we reported that high hydrostatic pressure (HHP) significantly improves post-thaw survival of frozen mouse and IVP bovine blastocysts, presumably from the induction of shock proteins (Pribenszky et al. 2005 Anim. Repr. Sci. 87, 143–150; 2005 Repr. Dom. Anim. 40, 338). We also have reported increased post-thaw survival of HHP-treated boar and bull semen (Pribenszky et al. 2004 Repr. Fert. Dev. 17, 199–200; 2005 Repr. Fert. Dev. 18, 162–163). We now report further data on the effect of HHP treatment on motility, viability, and fertility of frozen–thawed bull semen. HHP treatments were executed by a computer-controlled pressurizing device (Cryo-Innovation, Ltd., Budapest, Hungary). Semen of 21 bulls was diluted individually to a sperm concentration of 8 � 107 mL-1 with AndroMed� or Triladyl� extenders (MiniT�b, Tiefenbach, Germany). Diluted sperm was loaded into 0.25-mL straws at 25�C, and then divided into 2 groups: one group was treated with a HHP pulse defined and optimized earlier (30 MPa for 90 min); the other group was held at 5�C for the corresponding time. After HHP, treated samples also were placed at 5�C for 3–4 h. After equilibration at 5�C, samples were frozen (10 min at -110�C, and then plunged into LN2). Straws were thawed in a 35�C water bath for 30 s. Progressive motility was assessed by the CASA system (MiniT�b). Experiments were replicated twice for each bull. Paired t-test was used to analyze data. HHP treatment significantly increased the post-thaw motility of the frozen semen of the bull population examined. The mean of the differences was 21.14% (95% confidence interval: 13.56–28.72); P = 1.08 � 10-5. The post-thaw motility of 3, 7, and 5 of the 21 bulls increased by an additional 35–60%, 25–35%, and 10–25%, respectively; no effect was seen for 6 of the bulls. Four of the 7 bulls with low (3–19%) post-thaw motility were improved to the range of 43–70% by HHP treatment. Semen of 10 bulls was the subject of viability analysis individually. Sperm head, tail, and acrosome membrane integrity were evaluated with Kovacs-Foote staining (Kovacs and Foote 1992 Biotech. Histochem. 67, 119–124) and counting 300 sperm/sample. Paired t-test was used to analyze data. With HHP treatment, the proportion of the cells with intact tail, head, and acrosome increased significantly (P d 0.008). Eighty-two cows were inseminated with HHP-treated frozen–thawed semen; the 60 days non-return rate was 90.24%, whereas the 60 days non-return rate in the same population and time without treatment was 82.3% (n = 4789). Data were analyzed with the exact binomial test. HHP treatment significantly improved the non-return rate (P = 0.035; 95% confidence interval: 0.83–1.00). HHP treatment substantially increases the post-thaw semen quality of the bull population. Also, the semen of a proportion of bulls with very low semen freezability can be increased to the range where it can be frozen commercially. Further investigations are needed, including large-scale field trials incorporating the insemination of the otherwise low freezers and the biological background of the process. This work was supported by GVOP-TST050157 and Besamungsanstalt Klessheim.
A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2-4 h Giemsa staining at 25-40 degrees C is recommended for stallion semen.
Insemination of fresh boar semen is a widely used production tool in swine breeding and meat production. Although the success rates of routine insemination procedures are satisfactory, improvements are still needed to reduce production costs; moreover, the reduced litter size of gilts compared with sows still calls for further solutions. Sublethal high hydrostatic pressure (HHP), applied to fresh boar semen before cryopreservation, was reported to improve post-thaw motility and litter size. Proteomic studies revealed a treatment-related increase in different proteins that may contribute to increased fertility (Huang et al. 2008 Anim. Reprod. Sci., in press). Herein, we provide data showing how the same treatment influences the in vitro lifespan of semen stored at 15°C and fertility at routine fresh semen insemination. For in vitro evaluation, semen was collected from Seghers boars (n = 7), extended, cooled to room temperature, and then split into 2 groups. Semen in the treatment group was filled into sterile transfusion bags and treated with 300-bar pressure for 90 min at 25°C in a computer-controlled pressure device (Cryo-Innovation Ltd., Budapest, Hungary). After treatment, both groups were placed into a cooling thermostat set to 15°C. Total and progressive motility was assessed daily for 12 days by a computer-assisted semen analysis system. For in vivo evaluation, semen (boars, n = 14) was prepared, split, and treated as described above. Hungarian Large White × Hungarian Landrace sows (n = 103) were then inseminated with treated or nontreated semen. Inseminations were done in the routine production of 2 swine farms. For statistical analysis, a generalized linear mixed-effects model was used, with P < 0.05 regarded as significant. Results showed that the reduction of both total (TM) and progressive (PM) motility of the treated samples was significantly slower compared with control samples. On Day 5, the ratio of live (TM) to PM cells (control v. HHP treated) was as follows: TM, 55.4 v. 64.6%; PM, 36.6 v. 46.4%. On Day 11, the ratio of TM to PM cells was as follows: TM, 43 v. 53%; PM, 27 v. 31.4%. Mean pregnancy rates and litter weights were not different between the 2 groups (73 v. 74%; 16.34 v. 16.37 kg; HHP treated v. nontreated, respectively). However, the effect of treatment on the litter size was significant (12.4 v. 11.4; HHP treated v. nontreated). Moreover, there was a significant difference in treatment effects between the gilts and the sows: whereas treatment had no significant effect on the litter size of the sows (P = 0.47), the litter size of the gilts increased (P < 0.001), with a mean of 2.55 ± 0.83 (SE). This preliminary report shows that HHP-treated semen survived chilled storage better. The HHP treatment also increased the average live and total litter size achievable with fresh semen insemination, with litters not being different from the controls in weight, sex ratio, or stillbirths. The increased litter size was more evident in the case of the gilts. Further field trials are being conducted. This trial was supported by OMFB-00364/2007.
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