This study compared the milk protein, fat, lactose, total solids and amino acids (AA) profiles of Murrah buffalo, Nili-Ravi buffalo and crossbreed buffalo. Buffalo milk samples of Murrah (n ¼ 25), Nili-Ravi (n ¼ 20) and Triple-crossbred (n ¼ 23) were collected and milk protein, fat, lactose, total solids and 17 AA profiles were determined. Milk protein and total solids contents of Murrah buffalo were higher than those of Nili-Ravi buffalo (p < .05). The average milk protein, fat, lactose and total solids contents of all buffalo samples were 4.76, 7.31, 5.19 and 18.40 g/100 g of milk, respectively. The AA composition of all three kinds of milk samples was rich in glutamic acid (0.89-0.96 g/100 g of milk) and poor in cysteine (0.01-0.02 g/100 g of milk). Leucine, lysine, valine and isoleucine were the most prevalent essential amino acids; while glutamic acid, proline, aspartic acid and serine were the most prevalent non-essential amino acids in the three kinds of milk samples. Significant differences (p < .05) were observed for concentrations of Lys, Ile, Leu, Phe, Cys and His, while the other 11 amino acids had no significant differences (p > .05). Conclusively, the milk of Murrah buffalo, Nili-Ravi buffalo and crossbreed buffalo was all rich in glutamic acid while poor in cysteine contents. After crossbreeding, the milk production of Triplecrossbred buffalo was higher than the local Chinese buffalo and its milk composition was in between those of Murrah and Nili-Ravi.

The ruminal microbiota of 15 dairy buffalo was characterized using high‐throughput 16S rRNA gene amplicon sequencing. Results showed that Bacteroidetes was the dominant bacterial phylum in all rumen samples, followed by Firmicutes, Proteobacteria, Tenericutes and Verrucomicrobia. Butyrivibrio was positively correlated with average milk fat yield (R = 0.55; p = 0.03), average milk total solid yield (R = 0.56; p = 0.03) and standard milk yield (R = 0.52; p = 0.05). Acinetobacter were positively correlated with average milk protein yield (R = 0.56; p = 0.03), average milk total solid yield (R = 0.60; p = 0.02) and standard milk yield (R = 0.57; p = 0.03). Acetobacter was positively correlated with acetate (R = 0.63; p = 0.01), propionate content (R = 0.55; p = 0.03), butyrate content (R = 0.61; p = 0.02) and total VFA (R = 0.62; p = 0.01). The phyla Proteobacteria (R = 0.53; p = 0.04) and genus Prevotella (R = 0.52; p = 0.05) were positively correlated with butyrate content. Correlation analysis suggested that increased Butyrivibrio and Acinetobacter residing in the buffalo rumen could improve milk performance.

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Embryo-splitting technology provides an effective procedure for increasing the number of transferable embryos per donor, producing genetically identical offspring and facilitating embryo sexing. The ability to identify the sex of embryos before transfer will offer a reliable, economical and practical procedure for buffalo breeding. In this study, we have assessed the feasibility of production of offspring with controlled sex in buffalo by first comparing the effect of blastocyst quality on the viability of demi-embryos and then identifying the sex of a demi-embryo by multiplex-nested PCR before transfer into the recipient. In vitro-matured buffalo oocytes were fertilized by IVF and cultured to the blastocyst stage for 6 to 7 days as described by Lu et al. (2007 Anim. Reprod. Sci. 100, 192–196). These blastocysts were classified in terms of their developmental pattern and morphology on a scale of 1 to 3 grades as described by McEvoy et al. (1990 Theriogenology 33, 1245–1253). Blastocysts were split into 2 equal parts by a micromanipulation system. Viability of the resulting demi-embryos was confirmed by formation of a blastocoel cavity and definite inner cell mass after culture for 24 h. One of the zone-free demi-embryos derived from a grade-1 blastocyst was cultured in TCM 199 supplemented with 10% fetal bovine serum for another 2 h, then was transplanted to a spontaneous oestrous recipient. The other demi-embryo was used for sexing by multiplex-nested PCR (Fu et al. 2007 Theriogenology 68, 1211–1218). The results showed that grade-1 blastocysts yielded more viable demi-embryos than grade-2 and grade-3 blastocysts [P < 0.01; 73/92 (79.67%) vs 32/76 (47.05%) vs 26/94 (26.53%), respectively]. Transplantation of the presumed-Y demi-embryo derived from grade-1 blastocyst into a recipient resulted in the birth of a male buffalo calf. To the best of our knowledge, this is the first buffalo calf produced following embryo splitting and PCR sexing of the embryo at the blastocyst stage. Successful birth of the desired-sex offspring in the present study indicates the feasibility of using embryo splitting in combination with multiplex-nested PCR sexing to produce offspring of controlled sex in swamp buffalo. However, the quality of embryos before splitting was an important factor governing the in vitro development of viable demi-embryos. This study was supported by the Guangxi Science and Technology R&D Program (0626001-3-1, 0815008-2-4).