Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant.

Although the development of semen cryopreservation in the African elephants (Loxodonta africana) has been accomplished, effective procedures for cryopreservation of Asian elephant (Elephas maximus) spermatozoa have not been established. In the present study, we investigate the freezing methods for conservation of Asian elephant spermatozoa under field conditions and identify the most suitable freezing protocols which provide acceptable post-thaw semen quality. Semen was collected from two Asian elephant bulls (EM1 and EM2, 10 ejaculates from each bull) by manual manipulation and were assessed for volume, pH, sperm cell concentration, and progressive motility. Eight out of 20 ejaculates were of acceptable quality (progressive motility >/= 60%), and were used for cryopreservation studies. Semen were frozen in TEST + glycerol, TEST + DMSO, HEPT + glycerol, or HEPT + DMSO. The post-thaw progressive sperm motilities were assessed, and sperm cells were stained with PI and FITC-PNA for membrane and acrosomal integrity assessment using flow cytometry. Post-thaw progressive motility of spermatozoa (EM1: 42.0 +/- 4.3%; EM2: 26.0 +/- 17.3%) and the percentage of membrane and acrosome intact spermatozoa (EM1: 55.5 +/- 8.1%; EM2: 46.3 +/- 6.4%) cryopreserved in TEST + glycerol were significantly higher than (P < 0.05) those frozen in the other medium investigated choices for cryopreservation of Asian elephant spermatozoa. The data support the use of TEST + glycerol as an acceptable cryopreservation media of Asian elephant semen for the establishment of sperm banks.

Evaluation of the acrosome reaction and viability in buffalo spermatozoa using two staining methods: the effects of heparin and calcium ionophore A23187.

In this study, we investigated the effect of heparin and calcium ionophore A23187 on in vitro induction of buffalo sperm acrosome reaction (AR). Two methods for detection of the AR and viability were employed. Fluorescein isothiocyanate-conjugated Arachis hypogea agglutinin (FITC-PNA) was used as a vital stain in combination with ethidium homodimer-1 (EthD-1) to determine the acrosome status of viable spermatozoa. In another experiment, trypan blue replaced EthD-1 to differentiate live and dead spermatozoa having undergone AR. The results from the two methods were significantly correlated (r > 0.9). Four different staining patterns were found in both methods. The FITC-PNA intensely labels the acrosome region of acrosome-intact spermatozoa. EthD-1 and trypan blue stained red and blue at the post-acrosomal region of dead spermatozoa, respectively. Spermatozoa incubated with heparin showed a significant increase ( p < 0.05) in the percentage of live acrosome-reacted sperm after 30 min incubation period. This trend continued and was significantly different over the entire incubation period when compared with the control group at the same interval. In the ionophore-treated group, the proportion of changes in live acrosome-intact and live acrosome-reacted spermatozoa was statistically significantly different ( p < 0.001) when compared with those treated with heparin at the same interval. The AR occurred sooner and to a greater extent when incubated with the ionophore but at 5 h of incubation the percentage of false acrosomal reaction was significantly higher than those in the control and heparin-treated groups. The results in this study indicated that the in vitro induction of AR by heparin and calcium ionophore evaluated by both methods could be used to assess sperm fertilizing capacity for in vitro fertilization of this species.

Synthesis of bioadhesive lectin-HPMA copolymer-cyclosporin conjugates.

An amino group containing cyclosporin A (CsA) derivative has been synthesized and conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via an aromatic azo bond, which can be specifically cleaved by azoreductase activity in colon to release the drug for the treatment of colon diseases. Lectins, peanut (Arachis hypogea) agglutinin (PNA) and wheat germ agglutinin (WGA), have been conjugated to HPMA copolymer-CsA derivative conjugates (PCsA), respectively, to give bioadhesive conjugates. The PNA and WGA are the targeting proteins that can bind to diseased colon tissue and healthy tissue, respectively. There were on average four P(CsA) copolymer chains attached on one WGA molecule with a drug content of 16.0 wt % and five P(CsA) copolymer chains attached on one PNA molecule with a drug content of 11.5 wt %. The incubation of a P(CsA) copolymer with the rat cecal contents resulted in the cleavage of the azo bond and release of the cyclosporin derivative. The biological evaluation of the conjugates is under way.