Figure 4. Factors reducing in vitro fertilization success.

A) When pooled sperm samples from 5 males were held for 1 or 2 h on ice and used to fertilize eggs from freshly squeezed individual females (N = 10 in vitro trials/ time point), there was no difference in their fertilization success at 1, 4 and 24 h (P>0.05; ANOVA), suggesting that there was no male/male interactions or effect on holding the sperm that inhibited fertilization. The 1 h condition was considered the control condition, because it usually takes a while to collect the sperm and eggs prior to in vitro fertilization. B) To determine if dehydration was a factor affecting the eggs during fertilization, individual (N = 19) or pooled (N = 4) female eggs were held for 2 min in 40 µl of HBSS or kept dry, and then fertilized with pooled sperm (N = 10). There was no difference amongst the groups (P>0.05), suggesting that dehydration was not a factor in reducing fertilization success of individual or pooled eggs. C) To determine if holding time was a factor affecting fertilization success, a clutch of eggs from a single female was divided and either fertilized immediately or 5 min after being held dry or in HBSS. Fertilization success was affected by a holding time of 5 min, and (as shown in 4B) not by whether it was held dry or wet (P<0.05; ANOVA). The means were significantly different (P<0.05) between the groups that had different letters.