| Whether in vitro embryo production,somatic cell nuclear transfer or microinjection are used,the success of these technologies usually depends on the quality of retrieved oocytes.At present,most of the oocytes in embryo laboratories are obtained from Slaughterhouse ovaries(SLH),which can obtain a large number of available oocytes.However,the source of the donor for slaughter is unknown and the genealogy is unclear.To carry out genetic improvement or germplasm expansion,oocytes with clear genealogy and genetic background are required.For small ruminants such as goats and goat,Laparoscopic ovum pick up is the most suitable method because it can repeatedly,minimally invasive,and efficiently recover oocytes from living organisms.In this experiment,Saanen Dairy Goat dairy goats were used as experimental donors.The number of follicles that can be aspirated,the recovery rate of oocytes,the availability rate,and the number of oocytes per goat were used as indicators to optimize factors such as the collection needle,surgical pathway,donor age,number of oocytes collection times,and the dose of domestic FSH hormone stimulation during laparoscopic live oocytes collection;Using cleavage rate and blastocyst rate as indicators,in vitro embryo production was performed on oocytes collected in vivo.By exploring and optimizing the core processes and influencing factors of laparoscopic oocytes retrieval,the aim is to improve the efficiency of Laparoscopic ovum pick up(LOPU)and In vitro embryo production(IVEP),and establish a technical system for laparoscopic ovum pick up of small ruminants that meets the needs of China.The research results of this experiment are as follows:1.An experiment was conducted on the effect of oocyte collection needle types on oocytes collection efficiency in vitro ovaries: 18 G and 20 G oocyte collection needles had significantly higher oocyte availability than 23 G oocyte collection needles(61.02% vs 59.57%vs 46.55%;P<0.05);The diameter of the 18 G oocyte collecting needle is thick,which causes significant damage to the donor ovarian tissue.Therefore,the 20 G oocyte collecting needle was selected for subsequent experiments;2.An experiment was conducted on the effect of the length of the oocyte collecting needle on the efficiency of oocyte collection on the ovaries in vitro.A single chamber oocyte collecting needle(20 G-L)with a length of 35 cm and an oocyte collecting needle(20 G-S)with a transparent polyethylene tube connected to the end of the needle tip at 0.5 cm were used for oocytes collection on the ovaries of slaughterhouses.The results showed that 20 G-S had significantly higher oocyte availability than 20 G-L(78.08% vs 59.14%;P<0.05);3.The effect of 20 G-L and 20 G-S oocyte collection on oocyte collection efficiency was validated in vivo: there was no significant difference in oocyte recovery rate and number of oocytes per goat using 20 G-L and 20 G-S oocyte collection(57.59 ± 10.39% vs 66.46 ± 3.07%,4.75 ± 1.71 vs 4.50 ± 2.80;P>0.05);The availability of oocytes using 20 G-S oocyte collection needles was significantly higher than that of 20 G-L oocyte collection needles(76.61 ± 16.86%vs 50.12 ± 11.97%;P<0.05);4.To investigate the effect of surgical pathway on the efficiency of LOPU: There was no significant difference in oocyte recovery rate,availability rate,and number of oocytes per goat between the two groups using two and three pathways for LOPU(62.46 ± 18.47% vs 54.88 ±14.78%,72.22 ± 16.39% vs 75.56 ± 20.18%,4.83 ± 3.66 vs 3.50 ± 3.27;P>0.05);5.To investigate the effect of age on LOPU efficiency: LOPU was performed on young goat(6-9 months old)and adult goat,respectively.The number of visible follicles,number of aspirated follicles,and number of oocytes per goat in the young group were higher than those in the adult group(14.68 ± 5.57 vs 12.00 ± 6.96,10.00 ± 4.69 vs 7.83 ± 2.93,6.17 ± 3.06 vs5.00 ± 2.97;P>0.05),and the availability of oocytes in the adult group was higher than that in the young group(77.13 ± 20.39% vs 72.06 ± 18.07%;P>0.05),But the difference is not significant;6.To investigate the effect of repeated oocyte collection on the efficiency of LOPU,goat were subjected to 4 repetitions of LOPU: with the increase of oocyte collection times,the average number of visible follicles,number of aspiratable follicles,and number of oocytes per goat showed a decreasing trend;The available oocytes obtained from the first LOPU were higher than those obtained from subsequent rounds,but the difference was not significant(P>0.05);Compared with only one collection,there was no significant difference in the number of visible follicles,number of aspirated follicles,oocyte recovery rate,availability rate,and number of oocytes per goat obtained after four repetitions of LOPU(P>0.05);7.To investigate the effect of FSH dose on LOPU efficiency,experiments were conducted on different doses of FSH(260 IU,180 IU,130 IU): as the FSH dose decreased,small follicles(<2 mm)gradually increased,while large follicles(>5 mm)gradually decreased;The average number of follicles(≥ 2 mm and ≤ 5 mm)in the 180 IU group was higher than that in the other three groups,and the number of oocytes per goat was significantly higher than that in the control group(P<0.05);8.To investigate the effect of FSH treatment on the efficiency of IVEP,oocytes collected from the FSH treatment group and the control group were subjected to in vitro fertilization.There was no significant difference in cleavage rate and blastocyst rate between the two groups after in vitro fertilization(61.95 ± 5.74% vs 66.06 ± 6.95%,32.95 ± 3.34% vs 28.81 ± 4.24%;P>0.05);9.To investigate the effect of oocytes from different sources on the efficiency of IVEP,in vitro fertilization was performed on oocytes collected from LOPU and SLH.There was no significant difference in cleavage rate and blastocyst rate between the two groups of oocytes after in vitro fertilization(64.83 ± 6.59% vs 70.82 ± 2.87%,29.86 ± 4.16% vs 31.86 ± 2.06%;P>0.05);Immunofluorescence staining was performed on two groups of blastocysts,and the results showed that there was no significant difference in the total cell count and intracellular cell mass cell count between the two groups(102.17 ± 3.55 vs 100.83 ± 4.17,25.67 ± 1.21 vs24.50 ± 2.88;P>0.05);10.Vitrification was performed on the blastocysts of the LOPU group,followed by laparoscopic embryo transfer after resuscitation.A total of 4 goat were transplanted,with one goat pregnant.The research results indicate that the diameter and length of the oocytes collecting needle can significantly affect the quality of retrieved oocytes,and using 20G-S for oocytes collection has the best effect;The surgical pathway of live harvesting has no significant impact on the efficiency of live harvesting;There was no significant difference in the number of retrieved oocytes and high-quality oocytes between young and adult goat;Repeated oocytes collection for 4 times does not affect the total number of oocytes collected and the number of high-quality oocytes;The dosage of FSH can significantly affect the proportion of large and small follicles,and there is a significant difference in the number of follicles that can be aspirated.The FSH group with 180 IU has the best oocytes collection efficiency;The effect of FSH treatment on the efficiency of IVEP was not significant for donors before oocyte collection;The influence of oocyte origin on the efficiency of IVEP is not significant. |
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