Birth of the first mithun (Bos frontalis) calf through artificial insemination

Abstract

The study describes the standardization of a suitable semen cryopreservation protocol for the first time in mithun (Bos frontalis) and birth of the first mithun calf through artificial insemination. The semen samples were collected from adult bulls through the rectal massage method and cryopreserved in liquid nitrogen using tris-egg yolk-glycerol diluent. The diluted semen samples were packaged in 0.50 ml straws and kept at 58C for 4 h for equilibration. Following the equilibration, the straws were frozen into liquid nitrogen vapour for 10 min and then plunged into liquid nitrogen for storage. It was observed that the progressive motility (%) decreased significantly (P , 0.01) in cryopreserved semen (43.3 6 4.1) compared with fresh samples (76.6 6 3.3). The percentages of live spermatozoa (P , 0.01) and spermatozoa with intact acrosome (P , 0.05) also decreased significantly in cryopreserved semen (54.0 6 3.3 and 64.6 6 5.3) compared with fresh samples (79.3 6 2.6 and 85.3 6 1.8). Simultaneously, the total morphological abnormality (%) was found to be significantly (P , 0.01) higher in cryopreserved samples (15.46 6 2.68) than in fresh semen (3.85 6 0.63). A total of three mithun cows were inseminated using the cryopreserved semen. All the cows conceived following insemination and gave birth to healthy calves. The study revealed that mithun semen can be cryopreserved efficiently using tris-egg yolk-glycerol diluent, which can be further used for artificial insemination.

Effect of concentration and addition method of glycerol on the quality of cryopreserved mithun (Bos frontalis) spermatozoa

“…Semen samples were collected from the experimental animals through rectal massage method as described previously (Dhali et al., 2008). Briefly, seminal vesicles were massaged for 5 min followed by the gentle milking of ampullae for 3–5 min, which resulted in erection and ejaculation.…”

“…Following ejaculation, neat semen drops were collected into a graduated test tube with the help of a funnel. The samples exhibiting a fresh‐sample motility score of 3 or more were only processed further for freezing (Dhali et al., 2008).…”

“…The samples were diluted and frozen according to the previously described method after partial modifications (Dhali et al., 2008). Briefly, after collection, the samples were partially diluted by adding 1 ml of pre‐warmed (37 °C) Tris–egg yolk extender (Tris‐hydroxymethyl aminomethane, 200.6 m m ; sodium citrate dehydrate, 45.6 m m ; fructose, 55.5 m m ; egg yolk, 20%; Penicillium G sodium salt, 1000 IU/ml; dihydrostreptomycin, 1 mg/ml).…”

“…The sperm traits (progressive motility, livability, acrosomal integrity and morphological abnormalities) were determined after final dilution, 4h equilibration (4 °C) and warming (37 °C) and freeze‐thawing and warming (37 °C) according to the previously described methods (Dhali et al., 2008). In brief, progressive motility (total percentage of progressively motile spermatozoa) was subjectively determined to the nearest of 10% by analysing 4–5 fields of view of the sample placed on a pre‐warmed slide (37 °C) under a cover slip by two persons (magnification 400 ×).…”

“…Previously, we have demonstrated that mithun sperm could be stored satisfactorily in Tris–egg yolk extender for 36 h at refrigeration temperature (Karunakaran et al., 2007). Subsequently, we have cryopreserved mithun sperm in Tris–egg yolk extender with glycerol (5.6%, added in a single dose at 37 °C) as a cryoprotectant and produced the first ever mithun calf through AI using the frozen sperm (Dhali et al., 2008). Nevertheless, the post‐freeze‐thaw quality of mithun spermatozoa has been found inferior compared to cattle (Somfai et al., 2002; Thun et al., 2002; Nagy et al., 2004; Januskauskas et al., 2005), which suggests that further optimization of the freezing protocol should be attempted for improving the quality of frozen–thawed mithun sperm.…”