The successful establishment of live egg harvesting technology for water buffaloes is of great significance for accelerating the breeding and reproduction process of high-quality water buffaloes, and protecting excellent water buffalo germplasm resources. In the process of live egg retrieval, various factors need to be considered to assess the imaging effects on successful egg retrieval.

1. The influence of age and individual on the effectiveness of egg retrieval

According to the live egg collection results of yellow cattle, cows of all ages can be used as donors for live egg collection. Young cows (13-15 months old) and even prepubertal calves (6-9 months old) can serve as live egg donors without affecting the function of their ovaries or future pregnancy rates; However, the sexual maturity of water buffaloes is relatively late, and the ovaries of breeding cows are generally smaller, so there is less research in the industry on live egg collection from breeding female water buffaloes. At present, there are studies on live egg collection of young water buffalo through hormone treatment, and the Guangxi Water Buffalo Research Institute of the Chinese Academy of Agricultural Sciences has been conducting live egg collection of adult water buffalo for a long time. Huang Youjun et al. [4] reported that 42 river type milk buffaloes were divided into three groups based on age: Group A (3-7 years old), Group B (8-12 years old), and Group C (13-17 years old) for live egg collection, with two collections per week. The results showed that Group A obtained (4.35 ± 3.14) usable oocytes, which was significantly higher than Group B (2.47 ± 2.58) and Group C (2.36 ± 2.24). Female buffaloes aged 3-7 were more suitable for live egg collection, and there were also significant individual differences.

Imported live egg collection device to assist in collecting eggs from cows

2. The influence of sexual cycle and physiological status on egg retrieval efficiency

According to the operational mechanism of live egg collection, as long as the egg collection needle can puncture the ovary (follicle) through the top of the cow's vaginal vault, live egg collection can be carried out. Therefore, regardless of the day of the sexual cycle or the lethargic female water buffalo, even female water buffalo that are producing milk, dry milk, or pregnant can be used for live egg collection, only considering the efficiency of live egg collection or the genetic value of the donor cow. The recovery and division rates of oocytes in pregnant cows are lower than those in non pregnant cows. The number of follicles that can be harvested and the recovery rate in lactating cows are similar to those in dry milk cows, while the availability and division rates of oocytes in lactating cows are significantly higher than those in dry milk cows.

Generally, female water buffaloes in the estrus period have fewer follicles that can be harvested from their ovaries, and the texture of the ovaries is relatively hard. As a result, the ovaries of developing cows are generally smaller, and it is not suitable to collect eggs from both in vivo. In terms of the sexual cycle, although the number of follicles that can be harvested varies at different times of the cycle; But after the first egg collection, the available follicles on each ovary have been mostly collected, and the follicular phase tends to be consistent. The subsequent collection efficiency is basically the same. Therefore, for long-term batch live egg collection, there is no need to deliberately select which day of the cycle to perform live egg collection. What needs to be considered is the interval between live egg collection.

3. The effect of hormone treatment on egg retrieval efficiency

Exogenous hormones can promote the development of ovarian follicles, increase the number and quality of oocytes available for collection. Fifteen Mediterranean young female water buffaloes (5-9 months old) were treated with 2000IU of pregnant horse serum gonadotropin (PMSG) and 2000IU of human chorionic gonadotropin (hCG) before live egg collection. The results showed that hormone treatment did not increase the total number of follicles in the ovaries, but could increase the number of intermediate and above follicles and increase the number of available oocytes. Five swamp type estrus water buffaloes and six postpartum lactating water buffaloes were divided into a follicle stimulating hormone (FSH) treatment group and a control group to study the efficiency of in vivo egg collection. The results showed that the number of retrieved oocytes from the FSH treated estrus water buffaloes and postpartum lactating water buffaloes was (3.7 ± 2.7) and (5.9 ± 3.5), respectively, and the difference was not significant (P>0.05); The recovery numbers of oocytes in the control group of estrus water buffalo and postpartum lactating water buffalo were (1.4 ± 1.3) and (0.7 ± 0.8), respectively, and the difference was not significant (P>0.05). The application of FSH treatment can increase the number of recovered oocytes.

4. The influence of interval and duration of live egg retrieval on the effectiveness of egg retrieval

The use of B-mode ultrasound guidance for live egg collection in water buffaloes may cause some damage to the vaginal vault and ovaries, but the recovery is relatively fast and does not affect the normal occurrence and development of follicles. Collecting eggs once every 3 days is feasible, safe, and effective. As the number of egg collections increases, both the average number of retrieved eggs and the recovery rate tend to decrease. After 9 months of continuous live egg collection from 8 dry milk female water buffaloes, starting with 2 live egg collections per week and changing to 1 live egg collection per week after 68 days, the results showed that an average of 2.56 cumulus oocyte complexes (COCs) were obtained in the first 6 months, with a blastocyst rate of 16%, and an average of 1.57 COCs were obtained in the last 3 months, with a blastocyst rate of 0. After 6 months of live egg collection, the follicle bank reserve of the female water buffalo is basically depleted. Although oocytes can be collected in the future, they cannot develop into blastocysts due to poor quality.

5. The influence of vacuum pump negative pressure on egg retrieval efficiency

The availability of egg retrieval mainly depends on the number and morphology of cumulus cells attached to the egg It is generally believed that the vacuum negative pressure range required for live egg collection in yellow cattle is 4666.2-15998.4Pa, with a suitable liquid flow rate of 20-40mL per minute. A vacuum pressure of 13332Pa is the optimal negative pressure for egg suction, at which point the number of high-quality oocytes is highest. As the pressure increases, although the recovery rate increases; But it causes significant morphological damage to oocytes and their surrounding granulosa cells, resulting in a significant decrease in oocyte fertilization and blastocyst rates. When collecting live eggs from milk buffalo, the actual negative pressure used was between 4666.2 and 7999.2 Pa, and the results showed that the proportion of naked eggs in the collected oocytes was also relatively high. In summary, the vacuum pressure required for live egg retrieval depends on the instrument used (including the type of egg retrieval needle and catheter system) and the breed of cattle.

6. The impact of seasons on egg harvesting efficiency

The estrus of water buffalo has strong seasonality, which also affects the live egg collection efficiency of water buffalo. Pang Chunying et al. [7] collected live egg collection data from 2006 to 2008, and found that the average number of retrieved eggs per cow during spring live egg collection was (4.80 ± 1.77), with (2.77 ± 1.19) available eggs; The average number of retrieved eggs per cow in summer is (3.72 ± 1.58), with (2.11 ± 1.11) available eggs; The average number of retrieved eggs per cow in autumn and winter is (5.88 ± 2.84), and the number of available eggs is (3.56 ± 1.51), indicating that the effectiveness of live egg collection by water buffaloes is greatly affected by the season. The best live egg collection effect is in autumn and winter, followed by spring, and the worst in summer. The reason may be that during the peak season of estrus, there are more follicles developing on the buffalo ovaries, and a larger number of follicles can be collected. The average number of retrieved eggs per cow increases accordingly, but there is no significant difference in the availability of oocytes between different seasons (P>0.05).

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