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Separation of sperm from seminal plasma is required in most semen freezing procedures. Semen is typically subjected to centrifugation to concentrate sperm into a pellet and allow removal of the seminal plasma prior to dilution in freezing extender. Centrifugation is a relatively effective method to recover sperm, however, the process also causes considerable sperm damage. The use of a dense, inert, and isotonic solution as a cushion in the bottom of the centrifuge tube allows a greater centrifugation speed to be applied and results in greater sperm recovery. The aim of the present work was to evaluate the effects of this cushioned centrifugation technique on in vitro sperm viability and functionality. Sperm-rich fractions from 16 fertile boars were diluted and cooled to 15�C; then subsamples were centrifuged by one of two different techniques. A standard method (SM), 800 g for 10 min in 50-mL tubes (Westendorf et al. 1975 Dtsch. Tier�rztl. Wschr. 82, 261-267) and a cushioned method (CM), 1000 g for 20 min using 45 mL of diluted semen on 5 mL of an isotonic iodixanol solution (60% w/v gradient) were performed. Sperm samples were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378-391) to detect changes in lipid packing disorder of the plasma membrane. Another set of sperm samples was incubated in the presence of (0.7 �M) 22,72-dichlorodihydrofluorescein diacetate (Gadea et al. 2005 J. Androl. 26, 396-404) to estimate production of reactive oxygen species (ROS). A final set of sperm samples was stained with peanut aggultinin-fluorscein isothiocyanate (PNA-FITC) and propidium iodide to evaluate the acrosome reaction. All of these parameters were evaluated by flow cytometry before and after centrifugation. ANOVA analysis revealed that centrifugation altered lipid packing disorder and viability. Raw semen (RS) had a larger number of viable low lipid disorder sperm than centrifuged semen (RS = 86.9a vs. SM = 81.64b vs. CM = 80.6b, P < 0.01) and a decreased number of dead sperm cells (RS = 9.5a vs. SM = 15.0b vs. CM = 16.3b, P < 0.01). However, the cushioned and standard centrifugation methods yielded similar results for all the parameters measured. No significant differences were found for generation of ROS or in the number of sperm exhibiting the acrosome reaction. In conclusion, compared to the standard centrifugation method, this simple cushioned modification is a more efficient means of processing boar semen for freezing because significantly less sample losses are detected; also, it provides similar levels of sperm viability and functionality, and consequently a higher number of doses per ejaculation can be produced.

In beef cattle production under pasture-based tropical conditions, the minerals most likely to be lacking are Ca, P, Na, Co, Cu, I, Se, and Zn, all of which are required for optimal growth and reproduction. Phosphorous deficiency, in particular, is a major problem for grazing cattle in many tropical regions. On the other hand, reproductive infectious diseases are the greatest threat to the production and profitability of beef cattle herds. To overcome reproductive failure, the present study aimed to evaluate the effects of injectable mineral supplementation and vaccination against reproductive diseases on synchronised Nelore females for fixed-time AI. A total of 1361 females were enrolled, and blood was collected from a random population (n=150) to measure serum antibodies against Leptospira sp., infectious bovine rhinotracheitis, and bovine viral diarrhea, mineral status, and creatinine. With Day 0 as the beginning of the synchronisation programme and Day 10 as the day of fixed-time AI, females were randomly assigned as follows. In the control treatment (n=365), no additional treatment was administered. In the Vaccine treatment (n=314), at Days −21 and 0 females received a 5-mL subcutaneous injection of BovigenRepro (Virbac; inactivated vaccine containing infectious bovine rhinotracheitis 1 and 5; bovine viral diarrhea 1 and 2; Campylobacter fetus ssp. fetus, venerealis, and venerealis biotype intermedius; and Leptospira pomona, wolffi, hardjo prajitno, icterohaemorrhagiae, canicola, copenhageni, bratislava, and hardjo bovis in an adjuvant of 10% Al(OH)3 with Se). In the Suppl treatment (n=314), at Day 0 females received a 15-mL intramuscular injection of Fosfosal (Virbac; 100mL containing Na glycerophosphate (14g), monosodium phosphate (20.1g), copper chloride (0.4g), potassium chloride (0.6g), magnesium chloride (2.5g), and sodium selenite (0.24g). In the SupplVacc treatment (n=363), females received a 5-mL subcutaneous injection of the vaccine at Days −21 and 0 as well as a 5-mL intramuscular injection of Fosfosal at Day 0. Pregnancy was verified by transrectal ultrasound at Days 40 and 100. Body condition score was recorded on Days −21 and 40. Data were analysed using the GLIMMIX procedure of SAS (SAS University edition, SAS Institute Inc.). When significant, Tukey and least squares means tests were used to compare means. Serum analysis showed that 89% of the animals had seroprevalence to at least two diseases. Average blood P concentration was 7.2mg dL−1. Pregnancy was affected by Ca levels (pregnant=2.55mg dL−1; non-pregnant=2.33mg dL−1; P&lt;0.05) and creatinine levels (pregnant=5.85mg dL−1; non-pregnant=5.76mg dL−1; P&lt;0.05). The average BCS was 3.2 (1=skinny; 5=fat), and there was no effect of time (Days −21 to 40). Pregnancy verified by transrectal ultrasound at Day 40 for the Vaccine, Suppl, SupplVacc, and control treatments was 54, 52, 57, and 49% (P=0.11), respectively. There was a tendency of higher pregnancy verified by transrectal ultrasound at Day 90 for the SupplVacc group (55%) compared with the control group (45%; P&lt;0.1). There was no effect of pregnancy loss among groups that were vaccinated (control=6%, vaccine=5%, Suppl=4%, and SupplVacc=3%). Considering only pluriparous cows, there was an increase in pregnancy per AI in the SupplVacc group (59%) compared with the control group (51%; P&lt;0.05). Once females presented a very good BCS, the supplement effect was only an immunostimulant. Starting a breeding season with supplementation and vaccination can increase pregnancy rate and profitability for farmers.

Boar semen freezing procedures include the use of centrifugation to concentrate sperm and remove seminal plasma prior to dilution in freezing extender. The centrifugation techniques employed have necessarily been a compromise between the need to recover as many spermatozoa as possible after centrifugation and the damage caused by pelleting the sperm. The use of an inert, dense, and isotonic solution as a cushion in the bottom of the tube leads to the use of higher-speed centrifugation to ensure maximum sperm recovery. However, it is necessary to know the viability and functionality of the samples after the thawing process. The aim of this work was to evaluate the effect of cushion-technique centrifugation on the in vitro sperm viability and the penetrating capacity after thawing. Sperm-rich fractions from five fertile boars were diluted and cooled to 15°C before centrifugation. Two centrifugation regimes were used: 800g for 10 min called the “standard method” (SM) (Westendorf P etal. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267) and 1000g for 20 min on an iodixanol isotonic solution 60% w/v gradient (Sigma Chemical Co., St. Louis, MO, USA) called the “cushion method” (CM). Spermatozoa were diluted in lactose/egg-yolk extender, cooled to 5°C over 2 h and then frozen with glycerol and Equex by classic methodology (Westendorf P et al. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267). Frozen sperm samples were thawed in a circulating water bath at 38°C for 30 s. To detect increases in plasma membrane lipid packing disorder and viability, frozen-thawed samples of sperm were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378–391) and evaluated by flow cytometry. In vitro penetration ability was assessed using the homologous in vitro penetration (hIVP) test with immature oocytes (Gadea and Matas 2000 Theriogenology 54, 1343–1357). ANOVA analysis revealed that centrifugation by CM showed higher values of intact viable spermatozoa than SM centrifugation (60.21 v. 54.68%, P < 0.05). The in vitro penetration assay showed no differences in penetration rate or mean number of sperm penetrated per oocyte. However, significant boar and interaction effects were found (P < 0.01). These results indicated that different effects of the treatment were found for every boar. In conclusion, the cushioned centrifugation method gives a simple means of processing porcine semen for freezing more efficiently without loss of fertilizing capacity. This work was supported by AGL-2003-03144.