High-concentration bovine serum albumin enhances fertilization ability of cold-stored rat sperm

Abstract

Cold transport of the cauda epididymides is a useful technique for shipping laboratory rat sperm. Cold transport of rat sperm avoids potential risks of microbiological infection, animal escape or death, and animal welfare issues. Previously, we reported that a cold-storage solution containing dimethyl sulfoxide and quercetin maintained the fertility of cold-stored rat sperm. However, cold-stored rat sperm exhibited a decreased fertilization rate after 24-h storage. To recover the fertility of cold-stored sperm, we focused on the effects of bovine serum albumin (BSA), a cholesterol acceptor that induces sperm capacitation. We sought to determine the optimal concentration of BSA in fertilization medium based on the fertility of cold-stored rat sperm. High concentrations of BSA (40 mg/ml) enhanced the fertilization rate of cold-stored rat sperm and maintained sperm fertility for 144 h. Embryos derived from cold-stored and BSA-treated sperm normally developed into pups after embryo transfer. In summary, high BSA concentrations enhanced the fertility of cold-stored rat sperm and prolonged the storage period to 144 h, thereby expanding the transportable region for genetically engineered rats.

Keywords: Bovine serum albumin; Cold storage; In vitro fertilization; Rat; Sperm.

Fig. 1.

Effect of different BSA concentrations on cold-stored sperm fertility. The cauda epididymides were cold stored in Lifor with 15% DMSO and 200 µg/ml quercetin at 4°C for 24 h. After cold storage, sperm were cultured in mHTF at various BSA concentrations (0–60 mg/ml), and cumulus-oocytes complexes collected from female rats were introduced into the sperm suspension. The total fertilization rate was defined as the number of fertilized oocytes divided by the total number of oocytes, multiplied by 100 (A). The monospermic fertilization rate was defined as the number of monospermic oocytes (two pronuclei and sperm tail or sperm tail in the cytoplasm) divided by the total number of oocytes and multiplied by 100 (B). The polyspermic fertilization rate was defined as the number of polyspermic oocytes (three pronuclei and more than two sperm tails or more than two sperm tails in the cytoplasm) divided by the total number of oocytes and multiplied by 100 (C). Results are expressed as mean ± SD (n = 5–6, 154 females and 11 males). * P < 0.05 compared to 0 mg/ml. ** P < 0.05 compared to 4 mg/ml.

Fig. 2.

Effect of different concentrations of BSA on the motility of cold-stored sperm. The cauda epididymides were cold-stored in Lifor with DMSO and quercetin at 4°C for 24 h. Cold-stored sperm were cultured in mHTF at various BSA concentrations (0–60 mg/ml) for 2 h, and motility was analyzed using an IVOS Sperm Analyzer. The motility parameters were measured: motility (A), progressive motility (B), VAP (C), VSL (D), ALH (E), BCF (F), and STR (G). Results are expressed as mean ± SD (n = 3–5, 8 males). * P < 0.05 compared to 0 mg/ml.

Fig. 3.

Effect of storage periods on fertilization rates of cold-stored sperm incubated in 4 or 40 mg/ml BSA. Cauda epididymides were cold stored in Lifor containing DMSO and quercetin at 4°C for 0–144 h. After cold storage, the sperm were cultured in mHTF with various BSA concentrations (4–40 mg/ml), and cumulus-oocytes complexes collected from female rats were introduced into the sperm suspension. The total fertilization rate was calculated as the number of fertilized oocytes divided by the total number of oocytes, multiplied by 100 (A). The monospermic fertilization rate was calculated as the number of monospermic oocytes (two pronuclei and a sperm tail, or a sperm tail in the cytoplasm) divided by the total number of oocytes and multiplied by 100 (B). The polyspermic fertilization rate was calculated as the number of polyspermic oocytes (more than three pronuclei and more than two sperm tails, or more than two sperm tails in the cytoplasm) divided by the total number of oocytes and multiplied by 100 (C). Results are expressed as mean ± SD (n = 4–8, 90 females and 45 males). * P < 0.05, compared with 4 and 40 mg/ml BSA at each time point.

Fig. 4.

Live pups derived from in vitro fertilization using 96 hours cold-stored rat sperm. The fertilized oocytes were transferred to the oviducts of pseudo-pregnant female rats. Embryos developed normally into live pups 22–23 days after transfer.