Crossbreeding effect of double-muscled cattle on in vitro embryo development and quality

Abstract

Nowadays, several developing countries have started to breed double-muscled cattle to their autochthonous cattle to improve meat production. However, the developmental competence of the resultant crossbreeding embryos is unknown. The objective of this study was to evaluate the effect of crossbreeding double-muscled (Belgian Blue; BB) semen with beef (Limousin; LIM) and dairy (Holstein-Friesian; HF) derived oocytes on embryo development and quality, using purebred BB as a control (BB oocytes fertilized by BB sperm). A single ejaculate of a BB bull was evaluated by Computer Assisted Sperm Analysis before using for in vitro fertilization. Ovaries from each breed were collected at the local slaughterhouse (n = 1,720 oocytes). All statistical analyses were performed using R-core (P < 0.05). Embryo quality was evaluated via differential-apoptotic staining of day 8 blastocysts. Cleavage (48 h post insemination) and day 8 blastocyst rates were greater (P < 0.05) for LIM (82.9 ± 6 and 27 ± 4.3%, respectively) than for BB (69.8 ± 8.5 and 19.6 ± 3.1%, respectively) and HF (45.1 ± 10 and 12.3 ± 2.2%, respectively). Holstein-Friesian presented lower cleavage and day 8 blastocyst rates than BB (P < 0.05). Limousin blastocysts presented a higher number (P < 0.05) of inner cell mass cells (ICM; 68 ± 7.8) than HF (40.4 ± 8.2). In conclusion, crossbreeding double-muscled cattle by in vitro fertilization with LIM oocytes yielded better embryo compared with the purebred combination, while the combination with HF oocytes produced the lowest rate of blastocysts.

Introduction

Double-muscled cattle might be a valid alternative for crossbreeding autochthonous cattle in developing countries to improve meat production. Double-muscling in cattle is a genetic condition that occurs in several breeds such as Belgian Blue (BB), Piedmontese, and Marchigiana [1]. It has been reported that double muscling in BB cattle significantly increases carcass yield and meat tenderness [[2]], characteristics that are desirable in the meat industry. Some research groups uncovered that double-muscling is caused by mutations in the myostatin gene [3]. Among other functions, myostatin appears to play a role in the regulation of protein synthesis and glucose metabolism [4]. Although this condition results in improved meat quality and quantity, it also comes along with problems associated with reduced reproductive performance [5]. The sub-development of the hip bone (relative to the size of the animal) [6], the extended gestation period [7], and the higher birth weight of the offspring is associated higher risk of dystocia and perinatal calf mortality in double-muscled cows than non-double-muscled cows [8].

Fertility is a multifactorial trait that could be affected by many extrinsic factors such as estrus detection, timing of artificial insemination (AI) relative to ovulation, AI technique, reproductive pathologies, among others [9]. The use of an in vitro approach could help us to eliminate these extrinsic confounders to focus on the inputs of the oocyte and sperm on early embryonic development. A previous study reported more competent oocytes in double-muscled BB than in Holstein-Friesian (HF) dairy cows [10]. In contrast, semen characteristics are known to be markedly lower in BB than HF or crossbreed bulls [11]. The latter might be associated with smaller reproductive organ sizes and a not well defined scrotal neck in BB bulls in comparison to non-double muscled cattle breeds [3]. However, no study has been performed to associate the potential contribution of BB semen crossbred with oocytes derived from other breeds on their subsequent embryo development and quality.

We hypothesized that crossbreeding double-muscled semen with oocytes derived from distinct beef and dairy breeds, will affect their subsequent in vitro embryo assessments. Thus, the objective of this study was to evaluate the effects of in vitro crossbreeding of double-muscled (BB) sperm with beef (Limousin; LIM), dairy (HF) as well as with double-muscled purebred (BB) derived oocytes on blastocyst development and quality. The evaluation of blastocyst development included cleavage rate (CR), and day 7 and 8 embryonic rates. For blastocyst quality, we evaluated the total cell number (TCN), trophectoderm cells (TE), inner cell mass (ICM), ICM/TCN ratio, total apoptotic cells (AC), and AC/TCN ratio via differential/apoptotic staining of day 8 blastocysts. This could be considered as a first step to study the potential contribution of BB bulls in crossbreeding systems to maintain the benefit of meat production without an undesirable toll in fertility.