Abstracts

Dominant follicle removal by ultrasound guided transvaginal aspiration and superovulatory response in Nellore cows

(Remoção do folículo dominante por aspiração transvaginal guiada por ultra-som e reposta superovulatória em vacas Nelore)

A. Gradela^1*, C.R. Esper¹, S.P.M. Matos², J.A. Lanza², L.A.G. Deragon², R.M. Malheiros¹

¹Faculdade de Ciências Agrárias e Veterinárias – UNESP

14870-000 – Jaboticabal, SP

²Nova Índia Genética S.A. - Uberaba –MG

Recebido para publicação em 4 de agosto de 1999.

Colaboradores: J.C. Barbosa, I.L. Almeida Júnior, A.A.M. Rosa e Silva

E-.mail: agradela@zaz.com.br

*Endereço para correspondência: Rua Rafael A.S. Vidal, 2164 – Vila Elizabeth

13560-390 - São Carlos, SP

Financiamento: FAPESP

ABSTRACT

The present study analyzed the physiological significance of the dominant follicle (DF) and its removal on the superovulatory response of 42 Nellore cows and tested the hypothesis that the number of subordinate small follicles (3-8mm) could be used as a single criterion for the evaluation of the presence or absence of a DF in the ovaries. Ultrasonographic examinations were performed two days before the beginning of superovulatory treatment (D0) and the cows divided into three groups: group without DF (NDF), group with a DF (DF) and group with an aspirated DF (ADF). The superovulatory response of the donors did not differ between groups, but the rate of embryonic viability was higher in NDF (69.40%) and ADF (68.99%) groups compared to DF (48.54%). The number of unfertilized structures did not differ between NDF and DF or between NDF and ADF groups, but was higher in DF compared to ADF. It is concluded that, in Nellore cows, the number of subordinate follicles is a safe criterion for the evaluation of the presence of a DF in the ovaries, but errors could be avoided if only 4-8mm follicles were considered, and that the presence of a DF at the beginning of superovulation reduced the embryonic viability rate and its removal by transvaginal aspiration guided by ultrasound induced embryonic viability rates similar to that observed in the absence of a DF.

Keywords: Bovine, Nellore, dominant follicle aspiration, superovulatory response

RESUMO

O presente estudo analisou o significado fisiológico do folículo dominante (FD) e sua remoção na resposta superovulatória de 42 vacas Nelore e testou a hipótese de que o número de folículos subordinados pequenos (3-8mm) poderia ser usado como critério único para a avaliação da presença ou ausência de um FD nos ovários. Exames ultra-sonográficos foram realizados dois dias antes do início do tratamento superovulatório (D0) e as vacas foram divididas em três grupos: grupo sem FD (SFD), grupo com FD (FD) e grupo com um FD aspirado (FDA). A resposta superovulatória das doadoras não diferiu entre os grupos, mas a taxa de viabilidade embrionária foi maior nos grupos SFD (69,40%) e FDA (68,99%) grupos, comparados ao FD (48,54%). O número de estruturas não fertilizadas não diferiu entre os grupos SFD e FD nem entre SFD e FDA, mas foi maior no grupo FD comparados ao FDA. Pode-se concluir que, em vacas Nelore, a determinação do número de folículos subordinados é um critério seguro para a avaliação da presença ou ausência de um FD nos ovários, mas erros poderiam ser evitados se apenas folículos de 4-8mm fossem considerados, e que a presença de um FD no início da superovulação reduz a taxa de viabilidade embrionária e sua remoção por aspiração transvaginal guiada por ultra-som induziu taxas de viablidade embrionárias semelhantes àquelas observadas na ausência de um FD.

Palavras-chave: Bovino, Nelore, aspiração do folículo dominante, resposta superovulatória

INTRODUCTION

Because of the outstanding position of Brazil in the world cattle industry, the Nellore breed plays a very important role; however, further studies are needed to understand the factors that affect embryo production and viability in Nellore cows submitted to superovulation.

Differences in the superovulatory responses of Bos taurus and Bos indicus donor cows have been observed (Donaldson, 1984; Domingues et al., 1985). A negative effect of the presence of a dominant follicle at the beginning of superovulation on embryo production and viability in Bos taurus cows has also been reported (Pierson & Ginther, 1988; Rouillier et al., 1990; Guilbault et al., 1991; Bungartz & Niemann, 1994). According to Bungartz & Niemann (1994), transvaginal aspiration of the dominant follicle guided by ultrasound permits the removal of this suppressive effect. These investigators also observed that the number of subordinate follicles in one surge could be used as a criterion of dominance in a single ultrasonographic examination.

On the basis of these considerations, and of the absence of information in the literature, and in view of the importance of the Nellore breed, the present study was undertaken with the following objectives: (1) to investigate the significance of the dominant follicle in terms of follicle growth and the superovulatory response of these cows and (2) to test the hypothesis that the number of subordinate (3-8mm) follicles in a follicular growth wave can be used to detect a dominant follicle in a single ultrasound examination.

MATERIAL AND METHODS

The study was conducted at the Embryo Transfer Center of Nova India Genética, Uberaba, Minas Gerais, Brazil.

Nellore cows ranging in age from 3.0 to 13.1 years (mean, 6.6 years) were selected as embryo donors after presenting two regular estrous cycles and after the detection of a functional CL in the ovaries and of appropriate gynecological conditions. The donors were maintained on pasture, with appropriate mineral supplementation (mineral and common salt) supplied ad libitum and fed grass or ground sugar cane and a concentrate (commercial ration or corn, or citrus pulp) depending on the month of the year (june to december/1998).

Ultrasonographic examinations were performed with a Scanner 200 Vet apparatus [Pie Medical, Maastricht, Netherlands] equipped with a 5.0/7.5MHz transrectal transducer. All exams were registered (Vídeo Print P67, ECG) for later analysis by examiners who were not aware of the results of the superovulatory treatment.

The ovaries were examined 48h before the beginning of the superovulatory treatment (D0), on the day of the beginning of superovulation induction (D2) and on the day of embryo collection (D13). The criterion for the definition of a follicle as dominant was that adopted by Bungartz & Niemann (1994), which is based on the number of small subordinate follicles (3-8mm) present in both ovaries (>10 follicles = no dominant follicle, <10 follicles = presence of a DF). The donors were divided into three experimental groups: with no DF on D0 (NDF, N = 15), with the presence of a DF (DF, N = 15), and with an aspirated DF (ADF, N = 12). In ADF group, DF was aspirated (Bols et al., 1995) and the follicular fluid (FF) was stored for later analysis by radioimmunoassay (RIA).

Superovulation was induced 2d after the first ultrasonographic evaluation (D2) with 8 intramuscular injections of FSH [ Pluset, Serovet, Rome, Italy] administered twice-a-day in decreasing doses (morning/evening: 2.5ml/2.5ml, 2.1ml/2.1ml, 1.4ml/1.4ml, and 1.0ml/1.0ml, for a total dose of 350 IU Pluset) for four consecutive days. In the evening of the third day of superovulation (D4), luteolysis was induced with a single intramuscular injection of PGF_2a [Ciosin, Coopers, Brazil] (1.0mg), together with FSH. The cows were inseminated three to four times at intervals of 14, 20, 26 and 32h after the first visualization of superovulation estrus (D6) by the same technician, using frozen semen.

Seven days after the first artificial insemination (D13), the number of CL and of non-ovulated follicles (NOF) was determined and the retrieved structures (RS= recovered embryos, non-fertilized oocytes, degenerated structures) and transferable embryos (TE) were collected by the nonsurgical method (Newcomb et al., 1978) and evaluated (Boland et al., 1978; Lindner & Wright, 1983). After classification and "washing" in PBS with 10% SFB, the embryos were transferred by the surgical method to dully prepared recipient animals or stored frozen.

Intrafollicular P4 and E-17b concentrations were estimated by direct solid phase RIA (Coat-A-Count TKPG and Coat-A-Count TKE [Diagnostic Products Corporation, Los Angeles, CA, USA], respectively), according to manufacturer’s instructions in order to determine the E-17b :P4 ratio in FF. The hormones were not extracted and all follicular fluid samples were assayed at the same time for each hormone analyzed in order to reduce variation. Fixed FF volumes (25ml and 50ml for E-17b and P4 determination, respectively) were diluted 1:3 and 1:1 in a Zero Calibrator (A). The sensitivity of the P4 assay in FF was 0.08ng/ml and the intra-assay and interassay CV ranged from 3.50 to 13.20% and from 4.30 to 14.80%, respectively. For E-17b the sensitivity of the assay in FF was 0.02ng/ml and the intra-assay and interassay CV ranged from 4.00 to 7.00% and from 4.20 to 8.10%, respectively.

Data are reported as mean ± SEM. Tukey test was used for statistical analysis, with the level of significance at 0.05 (Steel & Torrie, 1980).

RESULTS

The criterion employed in the present study for the definition of dominant follicle (DF) was precise in 88% of the evaluations. Only five donors (12%) among those classified as having no DF presented an increase in the diameter of the largest follicle between D0 and D2 and were transferred from NDF to DF. Only one donor (2%) showed an increase in the second largest follicle after DF aspiration and was transferred from ADF to DF group. Among ADF donors the aspirated follicular fluid volume ranged from 0.30 to 1.00ml.

In NDF group, 20% of the donors started superovulatory treatment on day 8 (D8) of the estrous cycle, 13% on D9, 13% on D10, 34% on D11 and 20% on D12. In DF group, 53% started superovulation on D8, 0% on D9, 13% on D10, 7% on D11 and 27% on D12. In ADF group, 0% started superovulation on D8, 17% on D9, 17% on D10, 41% on D11 and 25% on D12. There was no correlation between the day when superovulation started and the presence or absence of a DF in the ovaries or the embryonic viability rates. There was no difference (P>0.05) between groups with respect to the number of collections to which each donor was submitted during her reproductive life (1.53±0.19 in NDF; 1.87±0.19 in DF and 2.08±0.22 in ADF).

The interval between PGF_2a administration and the manifestation of estrus did not differ (P>0.05) between groups (42.86 ± 2.43 h in NDF, 40.89 ± 2.43 h in DF and 43.17 ± 2.71 h in ADF).

The mean number of CL, NOF, RS and TE per donor did not differ (P>0.05) between groups (Table 1). Although the embryonic viability rate (TE/RS´ 100) did not differ between NDF (69.40%) and ADF (68.99%) groups, it was higher (P<0.05) in these groups than in DF group (48.54%) (Table 1). Of the total number of TE, 99% were blastocysts in NDF, 85% in DF and 96% in ADF groups.

The number of non-fertilized (NF) oocytes did not differ (P>0.05) between the groups NDF and DF and between NDF and ADF, but it was higher (P<0.05) in DF than in ADF group (Table 1). The rate of non-fertilized structures (NFR = NF/RS´ 100) did not differ (P>0.05) between groups NDF and DF and between NDF and ADF, but was higher (P<0.05) in DF than in ADF group. The number of degenerate structures (DS) did not differ (P>0.05) between the groups and the rate of degenerate structures (DSR = DS/RS´ 100) did not differ (P>0.05) between NDF and DF group but was higher (P<0.05) in ADF compared to the remaining groups (Table 1).

DISCUSSION

The criterion adopted in the present study for the evaluation of dominant follicle two days before the beginning of superovulatory treatment (Bungartz & Niemann, 1994) proved to be satisfactory and safe since the proportion of estradiol-17b :progesterone was higher than 1.0 (2.05 ± 0.16ng/ml) in all follicular fluids aspirated in the group with an aspirated dominant follicle, confirming physiological, and not simply morphological, dominance of these follicles (Stock et al., 1996).

The 12% error rate observed in the evaluations (non-dominant follicles whose diameter increased two days later) was probably due to the fact that follicles larger than 3mm might have regressed before reaching larger sizes, in contrast to follicles > 4mm (Ginther et al., 1997). On the basis of these considerations, it may infer that this type of error could have been avoided if only follicles measuring > 4mm in diameter had been considered. On the other hand, the increase in diameter of the second largest follicle after dominant follicle aspiration observed in one donor (2%) might have occurred because subordinate follicles might have responded to the FSH surge triggered by dominant follicle aspiration when the surge occurred during the first three days of dominant follicle growth, but not after five days (Ko et al., 1991; Adams et al., 1993). Thus, in this donor the second largest follicle became the dominant one.

According to data reported for Nellore cows and heifers (Figueiredo et al., 1997) and to the data obtained in the present study, although the superovulatory treatment can be started between the 8th and 12th day of the estrous cycle, Nellore donors with three surges of follicular development have a better probability of presenting a dominant follicle between days 11 and 12 of the cycle, whereas in animals with two surges this will occur more frequently between days 8 and 10.

The mean number of retrieved structures and transferable embryos detected in the present study were similar to those reported in the literature for Nellore cows (12.75 ± 1.80 and 5.60 ± 1.37, respectively; Gradela et. al., 1994). In contrast to the present study, increased numbers of transferable embryos (Huhtinen et al., 1992; Bungartz & Niemann, 1994) and retrieved structures (Gray et al., 1992) have been reported when superovulation is started in the absence of a dominant follicle. Although the superovulatory response and the number of recovered degenerated structures did not differ between the groups studied, the number of non-fertilized recovered structures was higher (P<0.05) in the group with a dominant follicle than in the group with an aspirated dominant follicle and the embryonic viability rate was lower (P<0.05) for the group with a dominant follicle at the beginning of superovulation (Table 1). These results disagree with those reported by others for Bos taurus cows (Ireland & Roche, 1987; Guilbault et al., 1991; Huhtinen et al., 1992; Bungartz & Niemann, 1994), which observed decreased superovulatory responses when the superovulatory treatment was begun at the presence of dominant follicles.

The negative effect of the dominant follicle on the embryonic viability rate in the group with a dominant follicle probably occurred due to its premature ovulation and formation of a small CL which impaired the ovulation of other follicles (Stock et al., 1996). The high plasma progesterone concentrations produced altered the normal secretory and motility patterns of the oviducts (Hawk et al., 1988) and caused a change in the sperm transport (Crisman et al., 1980) impairing the fertilization.

The beneficial effect of dominant follicle aspiration two days before the beginning of superovulation was confirmed in the group with an aspirated dominant follicle, in which the embryonic viability rate was similar to that for the group without dominant follicle. The absence of a significant difference between the superovulatory response of the donors in the present study is based on the assumption of a high homogeneity of the responses to gonadotropin stimulation, in contrast to previous reports about European (Callesen et al., 1988) and Zebu (Gradela et al., 1996) cows.

In conclusion, the present results strongly suggest that, in Nellore cows: 1) the detection of the dominant follicle on the basis of the number of subordinate follicles equal or more than 3mm in diameter two days before the beginning of superovulatory treatment was satisfactory, but errors could be avoided if only 4-8mm follicles were considered, 2) the presence of a dominant follicle did not impaired the superovulatory response in Nellore cows, but it impaired the embryonic viability rate of the recovered embryos and 3) the dominant follicle removal led to embryonic viability rates similar to those of animals submitted to superovulation in the absence of a dominant follicle.

ACKNOWLEDGMENTS

The authors are grateful to FAPESP, Brazil, for financial support; to Dalmares for laboratory assistance and to Nova Índia Genética S.A. for providing the cows.

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