Investigation and Preventive Measures of Fish Parasites in Huaihua City

Guangzhong HUANG Xu YANG Hui HU

Abstract［Objectives］ This study was conducted to investigate the effects of lamb age and in vitro culture system of oocytes on the results of juvenile in vitro embryo transfer (JIVET).

［Methods］ Ten Dorper×smalltailed Han lambs aged 5 to 10 weeks were induced to superovulate via i.p. injection of pregnant mares serum gonadotropin (PMSG). The oocytes were matured in basal maturation solution or modified maturation solution, which was prepared by adding 200 μmol/L cysteine to the basal maturation solution. Then, the oocytes were fertilized in fertilization medium I containing 2% estrus sheep serum (ESS) or fertilization medium II containing 3 mg/ml bull serum albumin (BSA). Finally, the number of oocytes, oocyte maturation rate and cleavage rate of the lambs of different ages were determined.

［Results］ The average number of oocytes recovered per lamb was (111.00±16.97), (139.50±28.99), (108.50±17.68) and (42.00±11.31) for 5, 7, 8 and 10weekold Dorper×smalltailed Han lambs, respectively. The number of oocytes obtained from 5, 7 and 8weekold lambs was significantly higher than that from 10weekold lambs (P＜0.05), but there was no significant difference among 5, 7 and 8weekold lambs (P＞0.05). The maturation rate of oocytes cultured in modified maturation solution was 3.64% higher than that in basal maturation solution. The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II (P＜0.01).

［Conclusions］ The results of JIVET can be improved by harvesting oocytes from lambs aged 5-8 weeks, adding a certain amount of cysteine into oocyte maturation solution, and a certain amount of ESS into fertilization medium.

Key wordsJuvenile lambs; Age; Oocytes; In vitro culture system; JIVET; Maturation rate; Cleavage rate

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Received: October 8, 2018Accepted: November 12, 2018

Supported by Special Fund for National Hair Sheep Industrial Technology System (CARS3924); Science and Technology Development Program of Shanxi Province ( 201203110241); Science and Technology Innovation Team Project of Shanxi Province (201705D13102820); Financial Support of Agriculture of Shanxi Province (NYGX201503); Talent Project for Science and Technology Development in Outlaying Poor Areas, Frontier Ethnic Minority Areas and Old Revolutionary Base Areas of Shanxi Province, China (2017Sy128).

Yangyi MAO (1960- ), male, P. R. China, professor, devoted to the research about sheep genetics and breeding, Email: mao7094728@126.com.

* Corresponding author. Email: lhd2638@126.com.

Juvenile in vitro embryo transfer (JIVET) is a powerful technology to produce offspring and reduce the generation intervals of livestock via superovulation of young animals, in vitro maturation of oocytes and embryo transplantation. This method was first developed and reported by the South Australian Research & Development Institute (SARDI), and has now become a research topic in the fields of embryo biology and livestock reproduction［1-5］. JIVET technology has so far been studied experimentally in Suffolk sheep, Liaoning cashmere goat, Dorset Horn sheep and smalltailed Han sheep［6-12］. However, there are still some problems that need to be resolved in JIVET. For example, there is no standard procedure for superovulation of juvenile animals, the results of superovulation are quite variable among the lambs of different ages, some of the oocytes have poor ability to mature or to develop into blastocyst stage in vitro. Numerous studies have reported the effects of lamb age, hormone combinations, injection time and dose on superovulation during JIVET treatment［10, 13］. To simulate the in vivo conditions for oocyte maturation, fertilization and embryo development, gonadotropins, antioxidants, epidermal growth factor (EGF), and estrus sheep serum (ESS), bovine serum albumin (BSA), amino acids and other substances are added into in vitro oocyte culture medium, to improve the results of JIVET technology［14-15］. The present study was conducted to investigate the effects of lamb age, addition of cysteine into oocyte maturation solution, addition of ESS or BSA into fertilization medium on JIVET, and to provide a theoretical basis for optimizing JIVET system.

Materials and Methods

Animals

Dorper rams, Dorper×smalltailed Han lambs aged 10 weeks and recipient ewes were provided by the sheep farm of Jinzhong Linshan Cooperative. Dorper sheeps semen frozen in pellet and ESS were prepared by the Laboratory of Animal Genetic Engineering of Institute of Animal Husbandry and Veterinary, Shanxi Academy of Agricultural Sciences.

Hormones and reagents

Folliclestimulating hormone (FSH), luteinizing hormone (LH), pregnant mares serum gonadotropin (PMSG), 17βestradiol (17βE2) and progesterone vaginal suppository (EaziBreed CIDR) were all purchased from Beijing Luxin Agriculture and Animal Husbandry Technology Co., Ltd.

Medium M199, cysteine, BSA, essential amino acids (EAAs), nonessential amino acids (NEAAs), and chemical reagents such as CaCl2?2H2O and NaHCO3 were purchased from Sigma.

Instruments

The main instruments used in this study included Olympus Szx10 stereo microscope, Olympus IX71 inverted microscope and Sanyo MCO5AC CO2 incubator. Surgical supplies including scalpels, scissors and hemostats were all purchased from Shanxi Medical Equipment Company.

Culture media

Oocyte collection medium consisted of 20 mmol/L Hepes, 2% ESS and 10 mg/ml heparin sodium. Basal oocyte maturation medium contained 20% ESS, 10 μg/ml FSH, 10 μg/ml LH and 1 μg/ml 17βE2. Modified oocyte maturation medium was prepared by adding 200 μmol/L cysteine to the basic oocyte maturation medium. Sperm capacitating medium (CM) was synthetic oviduct fluid (SOF, containing 20 μg/ml heparin sodium). Fertilization medium I was SOF supplemented with 2% ESS. Fertilization medium II was SOF supplemented with 3 mg/ml BSA. Fertilized eggs were cultured in SOF supplemented with 8 mg/ml BSA, 2% EAA and 1% NEAA.

Time and location

In vitro maturation and fertilization of oocytes was carried out in the Laboratory of Animal Genetic Engineering of the Institute of Animal Husbandry and Veterinary Medicine, Shanxi Academy of Agricultural Sciences from May 5 to 23, 2014, and the fertilized eggs were transplanted into recipient ewes at the sheep farm of Jinzhong Linshan Cooperative.

Methods

Superovulation and oocyte collection

Ten Dorper×smalltailed Han lambs aged 5 to 10 weeks were induced to superovulate via i.p. injection of 300 to 350 IU pregnant mares serum gonadotropin (PMSG) per lamb as previously described［1, 3］. And oocytes were recovered from ovaries according to Bais method［15］.

Oocyte maturation and fertilization

According to Bai［15］, all GradeA and GradeB oocytes were randomly and equally divided into two copies, one were cultured for 24 h in basal oocyte maturation medium, and the other in modified oocyte maturation medium. The oocytes were considered to be mature when the cumulus cells spread out or the first polar body was released. Then, the matured oocytes cultured in each oocyte maturation medium were randomly and equally divided into two copies, one were fertilized in fertilization medium I, and the other in fertilization medium II. The fertilized eggs were further cultured in embryo culture medium for about 20 h, observed under a microscope to calculate cleavage rate. After another 4 to 5 d of culture, the blastocysts of in each dish were calculated to calculate blastocyst formation rate.

Transplantation of embryos

The 2 to 8cell embryos were transplanted into recipient ewes according to Bais method［15］.

Data processing and analysis

The experimental data were sorted with Microsoft Excel, and analyzed with ttest and chisquare test for statistical significance.

Results and Analysis

Superovulation of 5to 10week Dorper×smalltailed Han sheep

The oocytes recovered from eight 5 to 10weekold lambs were shown in Table 1. The development and growth of their ovaries and follicles were shown in Fig. 1.

Table 1Oocytes recovered from each superovulated ewe lamb

Lamb No.Age∥weekTotal number of oocytesNumber of available oocytesAverage number of available oocytes

15123116111.00 a±16.97

259996

37119115139.50 a±28.99

47160149

58121114108.50 a±17.68

689694

710505042.00 b±11.31

8103434

Total802768

No. 1 and No. 2 are twin lambs; different lower case letters (a, b) within the column represent significant difference between means (P<0.05).

As can be seen from Table 1, a total of 768 available oocytes of Grade A and Grade B were obtained from the eight superovulated ewe lambs. The number of available oocytes of 5, 7, 8weekold lambs was significantly higher than that of 10weekold lambs (P<0.05), while there was no significant difference in number of available oocytes among 5, 7 and 8weekold lambs (P>0.05).

Effects of culture medium on in vitro maturation of oocytes

In vitro maturation of the 768 GradeA and GradeB oocytes in different culture media were shown in Table 2. The maturation rate of oocytes in modified oocyte maturation medium was 3.64% higher than that in basal oocyte maturation medium, and X2 test showed that the difference between the two them was not significant (P>0.05).

Table 2In vitro maturation of oocytes (Grade A and Grade B)

MediumTotal number of oocytesNumber of matured oocytesMutation rate∥%

Modified oocyte maturation medium38429276.00 a

Basic oocyte maturation medium38427872.40 a

Total76857074.22

The same lower case letter within the column indicates insignificant difference between treatments (P>0.05).

Effects of culture medium on in vitro development of fertilized oocytes

The development of fertilized embryos in different media was shown in Table 3, Fig. 2 and Fig. 3. As shown in Table 3, fertilization rate and blastocyst formation rate of matured oocytes in fertilization medium I were both higher than in fertilization medium II. And X2 test revealed that there was an extremely significant difference in cleavage rate between fertilization media I and II, but there was no significant difference in blastocyst formation rate among the four different treatments (P>0.05).

Table 3In vitro development of fertilized oocytes in different treatments

MediumFertilizationmediumNumber ofoocytesNumber ofcleaved embryosNumber ofblastocystsCleavagerate∥%Blastocystformation rate ∥%

Modified oocyte maturation mediumI146933163.70 Aa21.23

II146682646.58 Bc17.81

Basic oocyte maturation mediumI139832859.71 Ab20.14

II139602343.17 Bc16.55

Total570304108

The different upper case letters within column indicate extremely significant difference (P<0.01); different lower case letters indicate significant difference (P<0.05), and the same lower case letter indicate insignificant difference (P>0.05).

Agricultural Biotechnology2019

Transplantation of 2 to 8cell embryos

A total of 77 2 to 8cell embryos were produced, and nonsurgically transferred into the ampulla of the fallopian tube of 17 recipient ewes, acting as surrogate mothers. The results turned out that seven ewes got pregnant, and produced a total of 13 lambs. Among them, seven ewes gave birth to twins and one gave birth to singleton. The pregnancy rate of recipient ewes was 41.18%. on average, one JIVET lamb was produced using six 2 to 8cell embryos (Fig. 4).



Fig. 1Ovarian follicles of superovulated ewe lambs



Fig. 2In vitro cultured two to fourcell embryos



Fig. 3In vitro cultured blastocyst



Fig. 4Lambs produced via JIVET

Discussion

Effect of lamb age on JIVET technology

The young lambs were injected with gonadotropin to induce follicular development, and to produce a large number of oocytes, which is the basis for the establishment of JIVET system. It has been reported that lamb age is a critical factor influencing ovarian follicular development and JIVET results. In the study of Wang et al.［16］, gonadotropin was used to induce follicular development of Suffolk lambs of different ages, and the results showed that an average of 82.54 oocytes per lamb were produced by 5 to 6weekold lambs, and only 12.83 oocytes per lamb were obtained from 9weekold lambs. Our data showed that the number of available oocytes of 5, 7, 8weekold lambs was significantly higher than that of 10weekold lambs (P<0.05), which was basically consistent with previous studies［13, 16］. Generally, antral follicles first appear on fetal ovary at about 135 day of gestation. Then, this number continues to increase and reaches the maximum level 4 to 8 weeks after birth. The pituitary gland of lambs at this age is still incompletely developed, and the incidence of follicular atresia is limited［1］, so it is the best period to collect ovulated oocytes. Then, with the increase of lamb age and the development of the pituitary gland, lamb follicles gradually possess the ability to become dominant. Dominant follicles are more sensitive to folliclestimulating hormone (FSH), producing estradiol (E2) and inhibin, which inhibit the secretion of FSH from the pituitary gland. The nondominant follicles become atretic as FSH levels fall. Therefore, lambs aged 5-8 weeks are a preferred option of oocyte donors in JIVET system.

Effect of in vitro oocyte mature solution on JIVET technology

Oocyte maturation solution is an important factor influencing the results of JIVET. Bai［5］ reported that the oocyte maturation rate of young Mongolian Sheep was increased to 80.60% by 100 μmol/L βmercaptoethanol to oocyte maturation solution. Our data showed that the oocyte maturation rate of 5 to 10weekold lambs was improved from 72.40% in basal oocyte maturation solution to 76.04% in modified oocyte maturation solution, which was prepared by adding 200 μmol/L cysteine to the basal oocyte maturation solution. Cysteine is a substrate for the synthesis of glutathione (GSH), which is a tripeptide containing with a γamide bond and a sulfhydryl group, composed of glutamic acid, cysteine and glycine. GSH, present in every body cell, has antioxidant and detoxifying properties. And it protects the functional proteins and enzymes required for cell development, amino acid transport, protein synthesis, and DNA replication from being oxidized during oocyte development.

Effect of in vitro fertilization medium on JIVET technology

As one of the main culture media in JIVET system, fertilization medium should contain the nutrients required for sperm fertilization, oocyte growth and embryo development, maintain cell pH and osmotic pressure, and prevent oocyte zona pellucida from hardening, so that sperm can penetrate the zona pellucida to get to the oocyte. Guo et al.［17］ reported that the oocyte cleavage rate of Kazakh lambs was improved to 69.00%, and blastocyst formation rate to 17.50% in SOF fertilization medium (containing 20% ESS, 10 μg/ml heparin sodium). According to Wu et al.［18］, the oocyte cleavage rate of Xinjiang finewool lambs was 52.88% in SOF (containing 2% ESS) fertilization medium. Our data showed that the oocyte cleavage rate of Dorper×smalltailed Han lambs aged 5 to 10 weeks in fertilization medium I (containing 2% ESS) was very significantly higher than in fertilization medium II (containing 3 mg/ml BSA). ESS contains cell growth factors, reproductive hormones and proteins. Reproductive hormones (progesterone, estrogen) can bind to the progesterone receptor and estrogen receptor on sperm plasma membrane, and activate Ca2+ channel on sperm membrane, induce intracellular Ca2+ release and Ca2+ influx, and finally activate the enzymes involved in sperm capacitation and acrosome reaction. FSH, LH, E2, cell growth factors and proteins can promote oocyte cytoplasmic maturation, prevent oocyte zona pellucida hardening, conducive to subsequent spermegg binding, fertilization and embryo development. BSA is only a macromolecular protein in serum, and it is unstable. Therefore, the addition of a certain amount of ESS into the fertilization solution I is beneficial to the in vitro fertilization of lamb oocytes.

Conclusions

Our results showed that the number of oocytes obtained from 5 to 8weekold Dorper×smalltailed Han lambs was significantly higher than that from 10weekold lambs (P＜0.05). The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II (P＜0.01). The maturation rate of oocytes cultured in modified maturation solution was 3.64% higher than that in basal maturation solution. Our findings prove that it is possible to improve the results JIVET can be improved by recovering oocytes from lambs aged 5-8 weeks, adding a certain amount of cysteine into oocyte maturation solution, and a certain amount of ESS into fertilization medium.

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