SCIENCE

Animal Reproduction Science 47 ( 1997) 17 I- 180

Effects of using vasectomized bulls in artificial insemination practice on the reproductive efficiency of Italian buffalo cows
L. Zicarelli a,*, L. Esposito a, G. Campanile D.T. Armstrong b a, R. Di Palo a,

a Department Scienze Zootecniche. c;ia Delpino, I 80137, Naples, Italy b Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide, S.A.. Australia
Accepted 16 December 1996

Abstract The effects of the presence or absence of vasectomized male buffaloes on the reproductive efficiency of buffalo cows (n = 396) undergoing artificial insemination (AI) was studied on six farms owned and operated by a single consortium. Lactating animals were separated into two groups of various sizes on each farm and kept under semi-range conditions. Vasectomized bulls were present in one group at a bull/empty-cow ratio of 1:30. No bulls were present in the other group. Reproductive efficiency between the two groups over a period of 3.5 months was compared and evaluated on the basis of: 1) the number of spontaneous overt estruses associated with either feeble or intense signs of estrous behaviour; 2) the number of functional estrous cycles, i.e. estrous cycles with luteal phases defined as normal, based on specified progesterone concentrations in milk or blood plasma 8-10 days after estrus; 3) the number of consecutive functional estrous cycles in cases of induced estrus; and 41 pregnancy rate. Groups with bulls present demonstrated a significantly higher reproductive efficiency than groups without them. There was a higher incidence of spontaneous estrus (92 versus 69%; P < 0.01); spontaneous estrus of high intensity (62.2 versus 31.1%; P < 0.01); and higher incidence of functional estrous cycles following both spontaneous (65.8 versus 57.1%) and induced (77.0 versus 59.5%; P < 0.05) es&us. Exposure to vasectomised bulls also increased the incidence of consecutive functional estrous cycles (90.5 versus 68.1%; P < O.Ol>, and the pregnancy rate in cows inseminated at spontaneous (42.5 versus 18.9%; P < 0.01) or induced (5 1.1 versus 33.3%; P < 0.05) estrus. Overall pregnancy rate did not differ significantly between

* Corresponding

author. Tel.: 39 81 291663; Fax: 39 81 292981

0378-4320/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-4320(97)00008-O

172

L. Zicarelli et al./Animal Reproduction Science 47 (1997) 171-180

cows inseminated at induced or spontaneous rate per AI was higher in cows inseminated 18.9%). 0 1997 Elsevier Science B.V.

estrus, although in the absence of bulls, pregnancy at induced than at spontaneous e&us (33.3 versus

Ke.words: Buffalo; Vasectomized bull; Reproduction efficiency;

Artificial

insemination;

Estrous cycle

1. Introduction Es&us detection is difficult in buffalo because of the scarce behavioural signs which mark this event (Ohashi, 1994). It is also difficult to recognize buffalo cows in heat because of their uniform coat and because they cover themselves with mud. This detection problem has been reported by several authors from different countries where buffalo are mainly bred on small farms (Chauhan et al., 1976; Dane11et al., 1984; Drost et al., 1985). This problem is even more difficult in Italy, owing to the large number of head per farm and to the high labour cost, which leads to a high head/worker ratio producing major routine problems with AI use and being associated with low fertility. Progeny test inseminations performed in Italy during 1987-1988 produced a pregnancy rate of only 25.6% among 703 inseminated buffaloes (Villa and Fabbri, 1993). Previous research aimed to record the heat-ovulation interval in buffalo cows either with spontaneous or PGF2a induced estruses (Zicarelli et al., 1985; Zicarelli et al., 1988) showed that cows isolated from bulls for at least 6 months had dysfunctional estrous cycles and displayed no signs of estrous behaviour. Upon the reintroduction of a bull among these cows, their estrous cycles became functional again. Indeed, 55% of the cows were pregnant after 1 month, and 77% after 3 months. With this information, we planned an experiment to study the potential of bull presence as an aid in estrus detection in buffalo breeding as well as to validate the biostimulation effect as previously demonstrated in other livestock (Knight et al., 1978; Alberio et al., 1987; Nugent et al., 1988; Gifford et al., 1989; Naasz and Miller, 1990; Bums and Spitzer, 1992; Hornbuckle et al., 1995). The incidence of overt es&us, estrous behaviour signs, endocrinological competence of the luteal phase, and AI efficiency were used to test the hypothesis of male effect in buffalo. These parameters were compared either in spontaneous or PFG2 a-induced estruses. 2. Materials and methods 2.1. Management Mediterranean buffalo cows, distributed among six farms owned and operated by a single consortium in the province of Salerno, Italy, were used in the study. They were all kept under semi-range conditions and fed the same unifeed diet based on the following proportions per kg of dry matter: UFL, 0.8 (1 UFL = 1700 kcal net energy for lactation) (Vermorel, 1989); crude protein, 13.5-14.0%; crude fiber, 21%; Ca, 1.0%; P, 0.45%. The chemical analysis of the diet was performed according to A.S.P.A. protocols (Martillotti et al., 1987). On 15th October 1989 non-pregnant buffaloes were divided into two groups (B and W) on each farm. A vasectomized bull previously tested for libido, was then introduced

L. Zicarelli et al. /Animal

Reproduction Science 47 (19971 171-180

173

in each B group. The experiment started on 1st December. Those buffalo which had calved since 15th October were randomly allocated in W and B groups using a 2: 1 ratio. This ratio was imposed by space limitation of some of the farms, but was extended to all the farms to avoid unequal distribution of replications. There was always a maximum 1:30 male/female ratio in each B group as well as a homogeneous number of days postpartum and parity between W and B groups. 2.2. Estrus detection Estrus detection (Foote, 1974) was performed by trained technicians from 7:00 to 12:00 and from 13:00 to 17:00, this time representing the daylight interval between two milking periods. A further increase of this period would have required observations during night hours making the observation task difficult and without practical application for a future AI routine. When a cow displayed spontaneous, overtly evident estrous-behaviour, it was designated either as intense (marked by bellowing, homosexual mounting, and in B-groups. being sniffed, mounted, or serviced by the male), or feeble (marked by isolation, mucus discharge, stare and Flehmen signs). It was spray marked on its back with an identification number and given a rectal palpation. Cows were submitted for insemination on finding both a tonic uterus and a follicle larger than 1 cm. Spontaneous es&uses were categorized according to the above estrous behaviour signs and classified into two types: A and B. Type A was classified by behavioural signs involving only cows occurring in both B and W groups. Type B was defined by signs essentially involving detection by the bull and were present only in the B-groups. Type-A es&uses were further subdivided into three groupings: intense (A2), feeble (Al) and silent (AO). Type-B es&uses were subdivided into two groupings: bull served (B2) and bull sniffed (Bl). Table 1 shows: (i) the number of cows submitted to AI showing spontaneous es&us (overt and silent) and anestrus; and (ii) the number of cows which were submitted following estrus induction with PGF2 (Y (alfaprostol, Gabbrostim, Vetem, Italy; one dose was 8 mg). The PGF2 (Y was administered as follows: 1. as a single PGF2 (Y dose injected from 6 to 16 days after estrus (sub-group a); 2. as a double PGF2a dose injected 11 days apart in animals which previously had a spontaneous estrus (sub-group b); 3. as a single PGF2a dose administered to animals with a corpus luteum (CL) found at rectal palpation (sub-group c); and 4. as a single PGF2a dose administered to cows with a CL found at rectal palpation along with a high level of progesterone in the blood serum or milk sampled 24 h before injection (sub-group d). Cows in each of the four categories were artificially inseminated 60 h after they had received their last PGF2 (Y dose. AI was repeated every 24 h until ovulation was verified. In buffalo showing an overt es&us, a blood or milk sample was taken at the onset of estrus and repeated every 24 h until ovulation, as well as 8-10 days later. In buffalo submitted to estrus induction (a, b, c and d sub-groups), a milk or blood sample was taken at the time of PGF2 QI administration, or 24 h before (in the d sub-group) as well as 8-10 days later. By definition, animals in heat where those with a tonic uterus, an ovarian follicle larger than 1 cm as estimated by rectal palpation and with a progesterone

Table 1 Numerical groupings Total cows AI Category a Category b Category c Category d Spontaneous estruses Induced estruses

distribution

of B- and W-group

animals divided into estrus types and AI administration

Spontaneous estIUs cows

Anestrus cows

Induced estruses Al

B group 146 44 35 18 26 44 9 35 38 13 53 264 243 146 35 9 146 44 53 64 161 44 43 48 135

103 44

p & a ;E( S !k F \

53 b

Total

200

64 64

W group 53 17 5 8 13 53 17 5 21 8 28 36 22 16 41 79 22 14 36 72 53 22 16

53 22 a

$ 2 z 4 a % & S :: 3 R

16 b

41 132 91

Total

91

41

B+W 396 334 199 52 14 65 109 240 207

ti 2 S 2 S!

groups Total

291

105

I
at the time of AI or their ovulation had been mistakenly

aSpontaneous estrus cows included in A and B induced estrus categories. b Spontaneous estrus cows not submitted to AI at estrus detection because they either had already ovulated induced by rectal palpation or because uterine infection was diagnosed.

L. Zicarelli et al./Animal

Reproduction Science 47 (1997) 171-180

175

level less than 0.3 ng ml- in blood serum or 0.8 ng ml- in milk, respectively. A luteal phase was considered normal and/or a CL competent if it was associated with in blood serum or milk, respectively progesterone levels higher than 1.5 or 4.0ngml- (Zicarelli et al., 1982, 1988; Kamonpatana et al., 1983). 2.3. Anestrous cows and silent estruses

Cows not displaying estrous behaviour within a period of 30 days were palpated per rectum. If a follicle greater than 1 cm in diameter was found in the presence of a tonic uterus, a blood serum or milk sample was taken every day until ovulation, and then again 8-10 days after ovulation, These cows were classified as having a silent estrus when low progesterone concentrations were found in the sample taken at ovulation and high progesterone concentrations were found in the sample taken 8-10 days after ovulation. Upon determination of a silent estrus, cows were artificially inseminated every 24 h and palpated per rectum every 12 h until ovulation was verified. If, however, a CL was palpated along with a non-tonic uterus, estrus was induced with PGF2a. Cows meeting neither of these requirements for either insemination or estrus induction were recycled for another 30-days observation period. 2.4. Reproductive parameters

Pregnancy diagnosis was made at 45 days after the last AI administration and confirmed at 90 days by rectal palpation (P/AI). Other parameters included: (1) the number of spontaneous evident and non-evident (silent) es&uses (Table 1); (2) in cases of both spontaneous and induced animals, the number of functional estrous cycles @EC), i.e. es&uses in which 8-10 days after ovulation a high progesterone concentration was found; and (3) in cases of induced estrus, the consecutive FEC @ EC/competent CL), i.e. the number of FEC following PGF2 treatment in animals having a competent CL (CCL). 2.5. Statistical analysis An ANOVA test was used to analyse the number of es&uses per group. All other data were analyzed using a chi-squared test (Snedecor and Cochran, 1980) because: (1) the numerical distribution of lactating empty cows between farms was nearly equal; (2) the number of animals was maintained roughly in a 2:l ratio across B and W groups on each farm; and, (3) fertility rates did not differ significantly among farms.

3. Results 3.1. Effect of bull exposure on reproductive parameters during spontaneous estrus

3.1.1. Incidence and intensity of spontaneous estrus Spontaneous estrous behaviour was first observed, on average, at 108 and 12.5 days postpartum in B and W groups, respectively. However, these observations do not necessarily coincide with the onset of postpartum estrous behaviour because observa-

176

L. Zicarelli et al./Animal Reproduction Science 47 (1997) 171-180

Table 2 Effect of exposure to vasectomised per insemination (P/AI) Type of estrus

bulls on incidence

and type of spontaneous

estrus and on pregnancy

rate

Incidence of estrus (%) Ba W (18.6) A (11.1) A (18.9) (24.8) 41 (31.1) B 29 (22.01 B 21 (15.9) B 41 (31.1) b 132 (100)

Pregnancy B 51.4 a 38.1 40.0 38.5 45.4 42.5 A

rate P/AI W

(%)

A2, Al, B2, B 1,

intense feeble bull served bull sniffed

57 34 58 76

15.0 b 30.0 7.7 18.9 B

Silent estrus Anestrus Total

18 (5.9) A 64 (20.8) a 307 (100)

Groups: B, bull present; W, bull absent. Values with followed by different letters P < 0.01).

are significantly

different

(lower

case

P < 0.05;

upper

case

tions could not begin immediately after calving owing to B and W group formation requirements. Table 2 summarises the incidence of spontaneous estrus according to the type (intensity) of estrus which occurred in the two groups of cows. Overall, 243 spontaneous estruses were observed in 264 cows exposed to vasectomised bulls (Group B; 0.92 estruses per cow>, and 91 in 132 cows without bull exposure (Group W; 0.69 e&uses per cow) (P < 0.01). The number of animals in which no estrus was detected (anestrous animals) was higher in the W groups (31.1 versus 20.8%; P < 0.05). A higher proportion (62.2%) of Group B cows experienced an estrus of high intensity (types A2, B2 and B I> than among Group W cows (3 1.1%; P < 0.01). Conversely, the incidence of silent estruses was higher in Group W than in Group B cows (P < 0.01). Thus, the presence of bulls significantly increased both the overall incidence of overt estrus and the intensity of estrus, while decreasing the incidence of silent estrus. 3.1.2. Incidence of functional estrous cycles following spontaneous estrus The influence of bull exposure on incidence of functional estrous cycles @EC) is presented in Table 3. There was a tendency for exposure to bulls to increase the
Table 3 Effect of exposure to vasectomised bulls and type of spontaneous estrus on incidence cycles @EC) and on pregnancy rate per insemination in a FEC (P/AI: FEC) Type of estrus Incidence of FEC (%) B= Cow, intense Cow, feeble Bull, served Bull, sniffed Silent estrus Total 37/57 (65.7) 23/34 (67.6) 42/58 (72.4) 48/76 (63.2) lo/l8 (55.6) 160/243 (65.8) W 19/41 20/29 (46.3) (69.9) Pregnancy B 78.3 a 57.1 51.6 50.0 71.4 59.0 A of functional estrous

rate (P/AI: W

FEC) (%)

30.0 b 42.9 14.3 32.3 B

13/21 52/91

(61.9) (57.1)

a Groups: B, bull present: W, bulls absent. Values followed by different letters are significantly

different (lower case, P < 0.05; upper case, P < 0.01).

L. Zicarelli et al. /Animal Reproduction Science 47 11997) 171-180

171

percentage relationship

of estruses which resulted in functional estrous cycles. No significant was seen between intensity of estrus and incidence of FEC.

3.1.3. Effect of bull exposure on reproductive parameters following induced estrus The incidence of competent CL at the time of administration of PGF2a for estrus induction and of functional estrous cycles following estrus induction did not differ among the different methods of estrus induction. Exposure to bulls did not affect the proportion of cows exhibiting a competent CL at the time of estrus induction, but significantly increased the proportion of FEC per induced estrus (77.0 versus 59.5%; P < O.Ol), and the percentage of FEC per competent CL (90.5 versus 68.1%; P < 0.01). The interval between alfaprostol administration to ovulation was significantly shorter in B groups than in W groups (87.94 versus 98.84 h; P < 0.01; data not tabulated). 3.1.4. Pregnancy rates from AI at spontaneous and induced estrus In cows exhibiting spontaneous estrus, exposure to vasectomised bulls significantly increased overall pregnancy rate per AI (P/AI; P < 0.01; Table 2). This was evident both in cows exhibiting intense estrus (P < 0.05) and in cows exhibiting silent estrus, but not in those exhibiting feeble signs of es&us. However, in both W and B groups the fertility rate did not differ among the animals showing different signs of estrous behaviour, i.e. the fertility rate in B group did not differ between buffalo having overt estrus marked by a bull (Type B) or not (Type A) (39 versus 46%). Exposure to bulls similarly increased P/AI in cows in which estrus was induced (51 .l versus 33.3%; P < 0.05). Treatment effects on pregnancy rate per AI during a functional estrous cycle (P/AI: FEC) were similar to those of overall P/AI in cows expressing spontaneous es&us, with significantly greater rates observed in cows exposed to vasectomised bulls (P < 0.01) (Table 3). A similar tendency, although not statistically significant, was seen in cows in which estrus was induced. In cows in which es&us was induced, pregnancy rate per functional estrous cycle was enhanced by bull exposure in cows exhibiting two successive competent CL (P/AI: CCL) (62.7 versus 38.1%; P < 0.01).

70 60 .

66.4

Induced estms (P/AI)

sp0nmneOus

Induced QmJS (WAIFEC)

Fig. 1. Effect of exposure induced estrus.

to vasectomised

bulls on pregnancy

rate of buffaloes

from AI at spontaneous

and

178

L. Zicarelli et al./Animal

Reproduction Science 47 (1997) 171-180

The difference in fertility rates between B and W groups was even more pronounced during the February-March period, the beginning of the low breeding (anestrous) season for the Italian buffalo, in both spontaneous (57 versus 0%; P < 0.05) and induced (44.3 versus 22.1%; P < 0.05) e&uses (data not tabulated). Overall pregnancy rate per AI was greater in cows inseminated at induced estrus than in those inseminated at spontaneous estrus (Fig. l), whether exposed to bulls or not. A significant interaction occurred between effects of type of estrus (spontaneous versus induced) and exposure to bulls, in pregnancy rate per AI during a functional estrous cycle (P/AI: FEC). Thus, exposure to bulls resulted in higher P/AI: FEC in cows inseminated during spontaneous es&us, but not in cows inseminated during an induced estrus. The number of semen doses used to inseminate B- and W-group animals were approximately the same for each group regardless of whether the estrus was spontaneous (1.63 versus 1.73) or induced (1.66 versus 1.85). 4. Discussion The higher percentage of animals with obvious signs of estrous behaviour (A2, Bl and B2 types) together with the lower number of silent heat recorded in B versus W groups has demonstrated that the presence of a bull aids estrus detection in buffalo as reported in other species (Foote, 1974; Nugent et al., 1988; Naasz and Miller, 1990; Burns and Spitzer, 1992). In addition, bull exposure was associated with an increased pregnancy rate. This increase cannot merely be attributed to the bull ability to identify s animals with true estrus or the most appropriate time for AI. Only 55.1% (134 Type-B estruses in B-groups of 243 total es&uses in B groups) of all spontaneous evident estruses were detected by bulls, and B-group cows not detected by a bull (Table 2, Type A) had a pregnancy rate similar to those detected by a bull (Table 2, Type B): 46.3 versus 39.2%, respectively (data not tabulated). However, some bulls detected cows only after they had ovulated or in presence of high progesterone levels (bull estrus detection error of 9%; not shown in Tables). In addition, the difference in pregnancy rate between B and W groups was found also in case of induced estruses in which AI was administered at a scheduled time, independently from estrous behaviour signs, estrus detection and, hence, human error. Furthermore, a similar number of semen doses was used with animals included in B and W groups. Parameters other than pregnancy rate can be used to validate effects of biostimulation. In fact, in the case of induced e&uses, the AI/FEC ratio did not differ between B and W groups, but a higher fertility rate as P/AI/CCL was found in B group. Furthermore, in induced e&uses 9.5 versus 31.9% (complement of the FEC/CCL ratios) (P < 0.01) of buffalo cows in B and W groups having a competent CL at time of PGF2a administration, failed to develop another competent CL later. This last finding could explain the higher pregnancy rate recorded with spontaneous versus induced estruses under AI field application. Estrus induction is usually performed in the field upon observing only a small number of heats in the herd. Hence, fertility rates were influenced by the CCL and FEC incidence in the induced animals. In past studies (Zicarelli et al., 1988), the presence of a CL confirmed by palpation per rectum

L. Zicarelli et al./Animl

Reproduction Science 47 (1997) 171-180

179

at the time of PGF~(Y administration did not always ensure a functional estrous cycle. Indeed, only 84.6% of the spontaneous overt estrus animals chosen for induction (category A) and 66.2% of animals not exhibiting estrous behaviour (category C) (P < 0.05) a competent CL, despite confirmation of its presence by palpation per had rectum at the time induction was effected. Furthermore, when estrus induction by double PGF2 (Y administration (method B) was used, only 7 1.4% of the treated animals received the second dose of PGF2a in the presence of a CCL. In summary, a number of animals did not respond to induction, regardless of the method used. It is likely that short luteal phases are responsible for this lack of response (Baird, 1992; Garverick et al., 1992). However, in spontaneous estruses we do not have an explanation for the lack in statistical difference between B and W groups as regards the FEC incidence @EC/es&us) or for the different fertility rate between B and W groups when it is calculated as P/AI/FEC. In conclusion, the introduction of a vasectomized bull improves AI efficiency in buffalo cows having either spontaneous or induced es&uses. In our opinion it is difficult for buffaloes to adapt to breeding systems, such as AI, which do not take their ethological needs into account.

Acknowledgements The authors wish to thank Dr. Raffaele Boni for reviewing and editing this manuscript. They are grateful to the following farms: SAB, Iemma Gaetano, Eredi Salati-Ianniti, Carrozza, de Stefano, Desideri-Gaveglio for supplying and taking care of the animals. References
Alberio, R.H., Schiersmann, Cl., Carou, N., Me&e, J.. 1987. Effect of a teaser bull on ovarian and behavioural activity of suckling beef cows. Animal Reproduction Science 14, 263-272. Baird, D., 1992.. Luteotrophic control of the corpus luteum. Animal Reproduction Science 28, 95-102. Burns, P.D., Spitzer, J.C., 1992. Influence of biostimulation on reproduction in postpartum beef cows. Journal Animal Science 70, 358-362. Chauhan, F.S., Takkar, O.P., Tiwana, M.S., 1976. A note of incidence of estrus in buffaloes and cattle. Indian Veterinary Journal 53, 77. Danell, B., Gopakumar, N., Nair, M.C.S., Rajagopalan, K.. 1984. Heat symptoms and detection in Surti buffalo heifers. Indian Journal of Animal Reproduction 5, 1-7. Drost, M., Gripe, W.S., Richther, A.R., 1985. Es&us detection in buffaloes (Bubalus bubalis): use of an androgenized female. Buffalo Journal 1 (21, 159-161. Foote, R.H., 1974. Estrus detection and estrus detection aids. Journal of Dairy Science 58, 248-256. Garverick, H.A., Zollers, W.G., Jr., Smith, M.F., 1992. Mechanisms associated with corpus luteum lifespan in animals having normal or subnormal luteal function. Animal Reproduction Science 28, 111-124. Gifford, D.R.. D Occhio, M.J., Sharpe, P.H., Weatherly, T., Pittar, R.Y., Reeve, D.V., 1989. Return to cyclic ovarian activity following parturition in mature cows and first-calf beef heifers exposed to bulls. Animal Reproduction Science 19, 209-216. Hombuckle, T., Ot, R.S., Ohl, M.W., Zinn, G.M., Weston, P.G., Hixon. J.E., 1995. Effect of bull exposure on the cyclic activity of beef cows. Theriogenology 43, 411-418. Kamonpatana, M.. Pampai, R., Ngarmsuriyaroj, C., Srisakwattana, K., 1983. Plasma progesterone, oestrone and oestrone sulphate levels during the first half of gestation in swamp buffaloes. British Veterinary Journal 139. 256-261.

180

L. Zicarelli et al. /Animal Reproduction Science 47 (1997) 171-180

Knight, T.W., Peterson, A.J., Payne, E., 1978. The ovarian and hormonal response of the ewe to stimulation by the ram early in the breeding season. Theriogenology 10, 343. Martillotti, F., Antongiovanni, M., Rizzi, L., Santi, E., Bittante, G., 1987. Metodi di analisi per la valutazione degli alimenti di impiego zootecnico. Quademo Metodologico, no. 8. I.P.R.A., C.N.R., Rome. Naasz, C.D., Miller, H.L., 1990. Effect of bull exposure on postpartum interval and reproductive performance in beef cows. Canadian Journal of Animal Science 70, 537-542. Nugent, R.A., Notter, D.R., Beal, W.E., 1988. Effects of ewe breed and ram exposure on estrous behaviour in May and June. Journal Animal Science, 66, 1363-1370. Ohashi, O.M., 1994. Oestrus detection in buffalo cow. Buffalo Journal, Suppl. 2, 61-64. Snedecor, G.W., Co&ran, W.G., 1980. Statistical Methods, 17th ed. The Iowa State University Press, Iowa, pp. 73-74. Vermorel, M., 1989. Energy: the feed unit system. In: Jarrige, R. (ed.), Ruminant Nutrition, Recommended Allowances and Feed Tables. INRA, Paris. Villa, E., Fabbri, G., 1993. Results of A.I. in Italy for improvement schemes in buffalo. Proceedings of the International Symposium on Prospect of Buffalo Production in the Mediterranean and the Middle/East, Cairo, 9 November 1992, pp. 367-369. Zicarelli, L., Saviano, D., Coppola, A., Salvatore, M., 1982. Confront0 tra metodiche RIA per il dosaggio de1 progesterone nelle bovine e nelle bufale. Acta Medica Veterinaria 28, l-2. Zicarelli, L., Piccolo, V., Intrieri, F., 1985. 11 dosaggio de1 progesterone nel latte con metodo immunoenzimatico, per il controllo della fertilit nella specie bufalina. Proceedings of 6th National Congress on a Animal Production (ASPA). pp. 243-250. Zicarelli, L., Campanile, G., Infascelli, F., Esposito, L., 1988. Influenza de1 periodo, dell etl e della distanza dal parto sull anaestro primaverile della bufala. Rivista di Zootecnia e Veterinaria 16, 21-31.