64 COULD FROZEN–THAWED BOAR-SEMEN FERTILITY BE INCREASED WITH SEMINAL PLASMA ADDITION?

Abstract

Post-thawed addition of seminal plasma (SP) in equine cryopreserved semen increased the integrity of plasma and acrosomal membranes (Andrade et al. 2011 Reprod. Dom. Anim. 46, 682–686) and these possibly affect sperm lifespan, improving fertility (Garcia et al. 2010 Anim. Reprod. Sci. 119, 160–165). This study was conducted to verify the pregnancy (PR) and fertility rate (FR) of addition of homologous SP in thawed boar semen. First, SP collections were made with polled sperm rich-fraction. Semen was centrifuged (500 × g for 10 min) and supernatant was removed, centrifuged one more time (2500 × g for 30 min), vacuum filtered through membranes (0.22 μm), and stored at –80°C for future use. Samples collected to frozen were pooled and divided in 2 aliquots, control (cryopreserved with SP; CON) and cryopreserved without SP (WSP; SP was removed and discarded after centrifugation – 500 × g for 10 min). Samples were extended in freezing extender (Botupharma®) to a final concentration of 300 × 106 spermatozoa per milliliter, packaged in 0.5-mL straws, and frozen in an automatic system (TK Tecnologia em Congelação®) using a rate of –0.5°C min–1 until 5°C, –20°C min–1 until –120°C, and then immersed in LN (–196°C). Ten straws, from each treatment, were thawed in water bath (37°C/30 s) and extended in Beltsville thawing solution to obtain 1.5 × 109 sperm in 40 mL. The other 10 straws of WSP were thawed and extended in Beltsville thawing solution plus 10% (v:v) of SP to originate treatment TSP (thawed added of seminal plasma). Thirty-three (11 per treatment) gilts had synchronized ovulation with altrenogest (25 mg/5 mL, Regumate®) administration per 18 days. Following day after withdrawal the altrenogest was administered with an intramuscular injection of 600 IU of eCG (Novormon®) and 2.5 mg of pLH (Lutropin®-V) after 72 h; a single deep intrauterine insemination was made 36 h after. Five days after, females were slaughtered and embryos were collected (by oviducts flushed) and evaluated by esteromicroscopia. Fertility rate and PR were analysed by SAS program (SAS Institute Inc., 2010, Cary, NC, USA). Fertility rate was analysed by Mixed models, and treatment effects were analysed by orthogonal contrasts (C1: effect without SP = CW v. NC; C2: effect of post-thawed addition of SP = CP v. CW), and PR was evaluated by binary distribution with PROC GLIMMIX test. Fertility rate was not affected (P > 0.05) by cryopreservation of boar semen in presence or absence of SP nor by its addition in Beltsville thawing solution (10.58 ± 3.92, 9.57 ± 4.92, 21.29 ± 7.37 for CON, WSP, and TSP, respectively). Treatments did not influence (P > 0.05) PR (50.00, 36.36, 72.73 for CON, WSP, and TSP, respectively). Thus, neither SP addition in thawed boar semen nor cryopreservation with or without SP can be beneficial to PR and FR, in our experimental conditions. However, a numeric large difference was observed. Therefore, these results lead us to believe that SP have a potential to increase the fertility and pregnancy rate, and that can be verified in further studies, with more repetitions.Research was supported by Agroceres Multimix, Botupharma and FAPESP process 2013/08070-8 and 2011/23484-8.