Hormonal stimulation and oocyte maturational competence in prepuberal Mediterranean Italian buffaloes (Bubalus bubalis)

Theriogenology 57 (2002) 1877±1884

Hormonal stimulation and oocyte maturational competence in prepuberal Mediterranean Italian buffaloes (Bubalus bubalis) Giorgio Antonio Presiccea,*, Elena Maria Senatoreb, Giuseppe De Santisb, Romana Steccob, Giuseppina Maria Terzanob, Antonio Borgheseb, Guillermo Javier De Maurob a

ARSIAL, Centro Sperimentale per la Zootecnia, Via R. Lanciani, 38-00162 Rome, Italy b Istituto Sperimentale per la Zootecnia, Monterotondo, Rome, Italy Received 6 March 2001; accepted 13 September 2001

Abstract The objective of this study was to determine the best combined hormonal treatment to utilize in order to obtain a high number of good quality in vivo and in vitro matured oocytes from prepuberal Mediterranean Italian buffalo calves (Bubalus bubalis). Transvaginal ultrasound follicular aspiration was employed to recover oocytes from antral follicles. Fifteen barn housed buffalo calves, between 5 and 9 months of age were used in this study and randomly divided into control (Group A) and treated groups. A commercially available preparation of 2000 IU eCG was administered to animals in the treatment groups, followed by 2000 IU of hCG given either 12 h (Group B), or 24 h (Group C) before ovum pick up (OPU). From the time of administration of eCG treatments, the best timing for hCG administration before OPU was determined and integrated with the administration of 500 IU of FSHLH in a decreasing dosage protocol over 4 days (Group D). Expanded cumulus oocyte complexes (COCs) recovered from all groups were immediately ®xed for later aceto-orcein staining. All other COCs were processed for in vitro maturation using standard procedures and then ®xed and stained for assessment of nuclear maturation. Collectively, hormonal stimulation did not increase the number of ovarian antral follicles available compared to the control group (P > 0:05), but did result in higher output of medium (Group B: 9:8  7:1; Group C: 3:4  6:7; Group D: 15:6  4:9 versus Group A: 1:6  2:2) and large follicles (Group B: 44:8  22:9; Group C: 8:7  6:1; Group D: 70:2  10 versus Group A: 6:1  6:3). Administration of hCG 12 h before follicle aspiration proved to be the best strategy to obtain high numbers of immature and mature oocytes from antral follicles (P < 0:05; Group B: 70:8  12 and Group D: 82  12:6 versus Group A: 43:6  13:9 and Group C: 27:2  13:9). A signi®cantly higher number of expanded COCs was obtained from hormonally stimulated groups compared to the control group (P < 0:05; Group B: 28:7  16:8, Group C: * Corresponding author. Tel.: ‡39-6-862-73224; fax: ‡39-6-862-73249. E-mail address: [email protected] (G.A. Presicce).

0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 6 7 7 - 5

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16:3  5:9 and Group D: 27:1  16:9 versus Group A: 6:2  6). A higher oocyte maturational competence (P < 0:05) was found in Groups A, B and D (80:8  7:9, 87:5  8:2, and 86:5  4:3, respectively) compared to Group C (60  26:2). In conclusion, in prepuberal buffalo calves combined gonadotrophin stimulation protocols yielded higher numbers of medium to large size follicles compared to a control group. A high number of good quality oocytes were recovered by transvaginal ultrasound follicle aspiration, and a high rate of metaphase II progression was reached after in vivo and in vitro maturation. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Buffalo calves; OPU; In vitro±in vivo maturation; Hormonal stimulation

1. Introduction Newly developed reproductive technologies are being utilized to increase the genetic yearly gain in species of zootechnical importance. Within this framework, transvaginal oocyte recovery by puncture and aspiration of antral follicles has become a routine procedure in most laboratories where in vitro embryo production is part of the offered services to breeders. Prepuberal calves can offer an additional and earlier source of oocytes, and have been shown to be of practical use for genetic enhancement thanks to the possibility of reducing the generation interval [1±5]. The Mediterranean Italian buffalo (Bubalus bubalis) has a special regional zootechnical importance due to the features of its milk composition, leading to the production of high quality milk derived products of which mozzarella cheese is probably the best known worldwide [6,7]. An absence of regulation of milk production, and the higher price paid compared to the bovine for milk and derivatives, make the buffalo species a target of interest for entrepreneurs and for research groups interested in promoting and enhancing genetic potential. In the Bubalus species hormonal treatments for in vivo and in vitro embryo production have de®nitely been shown to be less effective than in bovine species [8±14], highlighting some species-speci®c inherent features. The use of ovum pick up (OPU) technique together with a re®nement of in vitro embryo production procedures may overcome these inef®ciencies and make possible a real and faster genetic improvement. Furthermore, within the multistep process leading to in vitro embryo development, the achievement of oocyte maturation is a key factor for embryo production [15±17]. In the buffalo there are some con¯icting reports about the time required for a full nuclear and cytoplasmic maturation [18,19]. The aim of this study was to evaluate the ef®ciency of three different combined hormonal treatments on follicle growth and oocyte quality, and to assess the rate of maturation progression immediately in recovered expanded COCs, and in immature COCs after in vitro maturation. 2. Materials and methods 2.1. Superovulatory treatments Fifteen barn housed prepuberal buffalo calves between 5 and 9 months of age were initially randomly divided into a control (Group A: untreated) and two treatment groups (each group n ˆ 5). Treatment groups received a subcutaneous progesterone ear implant

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Table 1 Superovulatory treatments Group

Days 0

3 a

B C De

C.I. C.I.a C.I.a

eCG eCG

4

5

6

7

8 b

!FSH-LHf

c

C.R. ‡ hCG (20:00 h) C.R.b ‡ hCG (08:00 h)c C.R.b ‡ hCG (20:00 h)c

OPU (08:00 h)d OPU (08:00 h)d OPU (08:00 h)d

a

Crestar implant. Crestar removal. c Time of hCG administration. d Time of OPU. e Same group previously subjected to OPU as control 3 weeks earlier. f Decreasing dosage of FSH-LH between 4 and 7 days. b

(Crestar, Intervet, Italy) followed 3 days later by 2000 IU i.m. of a commercially available preparation of eCG (Folligon, Intervet, Italy). Ear implant is beginning of treatmentÐDay 0. Four days after eCG administration, the Crestar implant was removed and a ®rst group (B) received 2000 IU hCG (1000 IU i.m. and 1000 IU i.v., Corulon, Intervet, Italy) 12 h before OPU, while the second group (C) received the same amount of hCG and route of administration 24 h before OPU. Three weeks after initial follicular aspiration, control animals were subjected to a superovulation protocol consisting of a commercial available preparation of 250 IU FSH and 250 IU LH (Pluset, BIO98, Italy) together with hCG (Group D). FSH-LH administration followed a twice daily decreasing dosage protocol for a total of 4 days (Days 4±7 in Table 1). In this last treatment group hCG was administered (same amount and route as previous eCG treatments) on the last day of hormone administration, together with Crestar removal, 12 h before follicle aspiration (Table 1). 2.2. Follicular aspiration Equipment for ultrasound oocyte retrieval consisted of an Aloka SSD-500 together with a 5 MHz sector scanner human vaginal probe (Aloka Co. Ltd., Tokyo, Japan) and 17.5gauge needle ®tting a proper metallic needle guide. Negative suction pressure was kept constant at 40 mmHg. Aspiration medium consisted of PBS with the addition of 10 IU/ ml of heparin. Buffalo calves were restrained in a squeeze chute and prepared for follicular aspiration as described by Presicce et al. [20] for bovine calves. 2.3. COCs processing Falcon tubes with the recovered follicular ¯uid were immediately sent to the lab for oocyte search. Immature oocytes were graded as A, 3 intact layers of compact granulosa cells; B, fewer than three layers; C, oocytes partially denuded; D, deprived of granulosa cells; DEG, degenerated oocytes characterized by granulosa cells with pycnotic nuclei or grossly visible altered oocyte cytoplasm; E, expanded COCs. Before ®xation treatment, COCs were also evaluated for granulosa cell color, pycnotic nuclei and oocyte cytoplasm color. Expanded COCs were judged matured, subjected to pipetting (0.02% hyaluronic

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acid) for removal of granulosa cells, and immediately ®xed for later aceto-orcein staining, while immature COCs were processed for in vitro maturation. Medium for in vitro maturation consisted of TCM 199, 10% fetal calf serum with the addition of 0.5 mg/ml LH, 5 mg/ml FSH and 1 mg/ml E2. Cumulus oocyte complexes (COCs) were grouped by quality, and 5±10 placed into 50 ml droplets covered by medical oil. A humidi®ed gas atmosphere of 5% CO2 in air at 39 8C was used for 22±23 h for in vitro maturation. After in vitro maturation, oocytes were denuded from granulosa cells by pipetting, and ®xed and stained as previously described for expanded COCs. 2.4. Statistical analysis Proportional data for oocyte quality and maturational competence were analyzed by ANOVA using the General Linear Model (SAS). 3. Results Total number of follicles ranged from 72 in Group B to 145 in Group D (P < 0:05), while remaining comparisons did not show statistical differences. As expected, a higher number of small follicles were obtained from control calves. Group C also recorded a similar high rate of small follicles. Higher numbers of medium to large size follicles were obtained from hormonally stimulated calves, with the exception of Group C, whose follicle production was more closely related to control Group A (Table 2). A smaller number of oocytes were recovered from Groups B, C and D when compared to control Group A. A similar number of Grade A and B COCs was observed among all groups, with the exception of Group C (P < 0:05). The number of expanded COCs was similar among treated groups and signi®cantly different from the control group (P < 0:05). Within the framework of this study, expanded COCs were considered to be of the same quality (intrafollicular maturation) as immature Grades A and B COCs, and when evaluated collectively treatment Groups B and D recorded the highest rate (P < 0:05) of good quality COCs (Table 3). Table 2 Follicles and follicle size among treatment groups Group

n; mean  S.D. Total follicles

A B C D

100; 20 72; 14.4 109; 21.8 145; 29

   

9.3de 7.6d 16.1de 4.1e

Small1 94; 92.2 32; 45.2 97; 87.8 20; 14

Medium2    

5.3d 27.4e 3.4d 9.1f

2; 1.6 8; 9.8 4; 3.4 23; 15.6

Values with different superscript (d, e, f) within columns differ (P < 0:05). 1 Respective follicle diameters: 3 mm. 2 Respective follicle diameter: 4±8 mm. 3 Respective follicle diameter: 9 mm.

   

Large3 2.2d 7.1ef 6.7de 4.9f

4; 6.1 32; 44.8 8; 8.7 102; 70.2

   

6.3d 22.9e 6.1d 10f

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Table 3 Oocyte quality among treatment groups Group

n; mean  S.D. A ‡ B1

Oocytes A B C D

87; 47; 77; 89;

84.5 65.4 63.3 62.3

   

11.7b 4c 19.3c 10.6c

34; 20; 8; 48;

37.3 42.1 10.8 54.9

E1    

12.5b 10.9b 9c 17.9b

6; 6.2 11; 28.7 10; 16.3 25; 27.1

A ‡ B ‡ E1    

6b 16.8c 5.9c 16.9c

40; 43.6 31; 70.8 18; 27.2 73; 82

   

13.9b 12c 13.9b 12.6c

Values with different superscript (b, c) within columns differ (P < 0:05). 1 Oocyte quality. See text for explanation of differences. Table 4 Oocyte maturational competence among treatment groups Group

n; mean  S.D. Metaphase II A ‡ B ‡ E1

A B C D

24; 27; 9; 60;

71.9 87.2 58.8 87.6

   

40.6cd 8.2d 25.9c 10.3d

Metaphase II total2 32; 80.8 28; 87.5 15; 60 70; 86.5

   

7.9c 8.2c 26.2d 4.3c

Values with different superscript (c, d) within columns differ (P < 0:05). 1 Maturational stage, respectively, in quality oocytes A ‡ B ‡ E. 2 Maturational stage, respectively, in the total number of usable oocytes (A ‡ B ‡ C ‡ E).

Due to the reduced number of expanded COCs recovered among control and treated groups, no signi®cant differences were highlighted when maturational competence was taken into account (not included in table) in this category of derived oocytes. When selected COCs (Grades A, B, and E) or the total number of usable COCs (Grades A, B, C, and E) were considered for maturational competence, then a signi®cant difference was evident between Group C and the remaining treatment groups (Table 4). 4. Discussion The data collected in this study reveal that, according to the in vitro criteria in use in the present study, high numbers of good quality expanded or immature COCs can be recovered from hormonally stimulated or untreated prepuberal buffalo calves by means of transvaginal ultrasound follicular aspiration, and that a high rate of nuclear maturation can be reached for successive in vitro fertilization. Despite their genetic inherently reduced follicle reservoir [21±23], compared to bovine calves, buffalo calves in this study have produced an equal [20] or higher number [24] of antral follicles either with or without exogenous hormonal stimulation. As in bovine calves, in control buffalo calves, clusters of very small follicles along the cortex of the ovaries were observed (personal observation). This made it impossible to count the follicles singly, although puncturing was effective in targeting most of them and recovering oocytes. As expected, in untreated calves most of the

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follicles were in the small size category. The largest follicles did not exceed 8±10 mm in diameter, as was the case also for the other treatment groups. The combined effect of eCG and hCG 24 h before OPU (Group C) in prepuberal buffalo calves resulted in the poorest results in terms of medium and large size follicle development compared to the other two treatment groups. One possible explanation may reside in a precocious follicle disruption due to an earlier hCG administration. Although some luteinic tissue was observed ultrasonically in all ®ve calves, it was not possible to ascertain with precision if more than one follicle had been involved. Despite the higher number of large follicles in the FSH-LH treated group (Group D), compared to the control Group A, oocyte recovery was, as expected, as low as in the other two hormonal treatment groups, very likely due to the rapidly collapsing follicular wall that may trap the COC. In this study though, a higher number of oocytes per animal and per treatment group were recovered compared to results reported by Techakumphu et al. [12]. Mucus discharge was observed among only the ®ve calves undergoing FSH-LH stimulation at the time of follicular aspiration, together with mounting behavior as previously reported for bovine calves [16,25,26]. A signi®cantly higher amount of hemorragic follicular ¯uid was aspirated from antral follicles in this group, making the search for COCs more dif®cult. When considering oocyte quality output based on the number of Grades A and B COCs, then Group C recorded a signi®cantly poorer response compared to the other groups, although this may not re¯ect a real biological difference. This difference may partly be attributed to the high individual variability (in one calf, no Grades A or B COCs were obtained) associated with a relatively small number of calves under scrutiny. The number of recovered Grade E COCs in treated groups was, as expected, similar among the groups, and signi®cantly higher than in the control group. When expanded COCs were included with Grades A and B COCs, then Groups B and D yielded the highest rate of good quality COCs. Maturational competence was found to be signi®cantly lower in Group C both when only selected oocytes and the total number of usable oocytes were taken into account. Overall, it seems that of the two available commercial gonadotrophin preparations, an equal amount of FSH and LH at the dosage given in this study, together with the support of hCG, results in better follicular growth rate and oocyte quality. A tentative speculation on this matter in the buffalo species may take into account a possible partial refractoriness to the eCG used, or alternatively, a less suitable administration protocol given the adopted long term single eCG administration. This partial reduced response to eCG compared to other preparations containing pure FSH, or in combination with various amounts of LH, has been described by several other authors in both cattle and buffaloes [8,10,12,26±28]. In this study, the additional use of hCG 12 h before follicular aspiration has signi®cantly increased the number of mature oocytes, and although a previous study has reported no bene®cial effects in terms of total in vitro embryo output when including in vivo matured oocytes [24], the combined administration seems ef®cient in yielding a high number of good quality COCs for successive in vitro embryo production. Although most of the oocytes recovered from untreated prepuberal calves, both in the bovine and buffalo species, derive from very small follicles, known to have a reduced developmental competence [29], in this study, based on morphological classi®cation, a similar rate of A and B grade COCs from Group A was recovered when compared to treatment Groups B and D, even though the latter groups recorded a higher number of medium to large size follicles. A traditional

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maturation time of 22±24 h was used for immature oocytes despite contrasting reports on the time required for buffalo immature oocytes to reach second metaphase [18,19,30], with resulting high rates of maturational occurrence. When Grades A and B COCs together with expanded COCs were considered, a similar maturational competence was observed among Groups A, B and D. In conclusion, a high rate of good quality COCs, morphologically competent to undergo in vitro fertilization after in vivo or in vitro maturation, were obtained from prepuberal buffalo calves. Hormonal stimulation protocols yielded the highest number of expanded COCs, and among these, FSH-LH treatment resulted in the highest number of oocytes progressing into metaphase II. Acknowledgements Part of this work was supported by project RAIZ. References [1] Armstrong DT, Holm P, Irvine B, Petersen BA, Stubbings RB, McLean D, et al. Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration. Theriogenology 1992;38:667±78. [2] Georges M, Massey JM. Velogenetics, or the synergistic use of marker assisted selection and germ-line manipulation. Theriogenology 1991;35:151±9. [3] Lohuis MM. Potential bene®ts of bovine embryo manipulation technologies to genetic improvement programs. Theriogenology 1995;43:51±60. [4] Madan ML, Singla SK, Chauhan MB, Manik RS. In vitro production and transfer of embryos in buffaloes. Theriogenology 1994;41:139±43. [5] Majerus V, De Roover R, Etienne D, Kaidi S, Massip A, Dessy F, et al. Embryo production by ovum pick up in unstimulated calves before and after puberty. Theriogenology 1999;52:1169±79. [6] Coppola S, Villani F, Coppola R, Parente E. Comparison of different starter systems for water buffalo mozzarella cheese manufacture. Lait 1990;70:411±23. [7] Spanghero M, Susmel P. Chemical composition and energy content of buffalo milk. J Dairy Res 1996;63:629±33. [8] Karaivanov C. Comparative studies on the superovulatory effect of PMSG and FSH in water buffalo (Bubalus bubalis). Theriogenology 1986;26:51±6. [9] Misra AK, Joshi BV, Kasiraj R, Sivaiah S, Rangareddi RS. Improved superovulatory regime for buffalo (Bubalus bubalis). Theriogenology 1991;35:245. [Abstract]. [10] Schallenberger E, Wagner HG, Papa R, Hartl P, Tenhumberg H. Endocrine evaluation of the induction of superovulation with PMSG in water buffalo (Bubalus bubalis). Theriogenology 1990;34:379±92. [11] Sharifuddin W, Jainudeen MR. Superovulation and non-surgical collection of ova in the water buffalo (Bubalus bubalis). In: Proceedings of the 10th International Congress on Animal Reproduction, vol. 4, Urbana, 1984. p. 240±42. [12] Techakumphu M, Lohachit C, Tantasuparak W, Intaramongkol C, Intaramongkol S. Ovarian responses and oocyte recovery in prepubertal swamp buffalo (Bubalus bubalis) calves after FSH or PMSG treatment. Theriogenology 2000;54:305±12. [13] Totey SM, Singh G, Taneja M, Pawshe CH, Talwar GP. In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis). J Reprod Fertil 1992;95:597±607. [14] Totey SM, Pawshe CH, Singh GP. Effect of bull and heparin and sperm concentrations on in vitro fertilization of buffalo (Bubalus bubalis) oocytes matured in vitro. Theriogenology 1993;39:887±98. [15] Arlotta T, Schwartz JL, First NL, Leibfreid-Rutledge ML. Aspects of follicle and oocyte stage that affect in vitro maturation and development of bovine oocytes. Theriogenology 1995;45:941±56.

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