Evaluation of seasonal variations of semen freezability in Leccese ram

Animal Reproduction Science 79 (2003) 93–102

Evaluation of seasonal variations of semen freezability in Leccese ram A.G. D’Alessandro∗ , G. Martemucci Dipartimento PRO.GE.S.A., Universita degli Studi di Bari, Via G. Amendola 165/A, 70126 Bari, Italy Received 2 July 2002; received in revised form 6 May 2003; accepted 6 May 2003

Abstract The experiment was carried out in Southern Italy (41◦ N latitude) to examine the effects of seasonal variations of semen freezability in Leccese ram. Semen from five rams, collected every 2 weeks for a whole year, was frozen in straws, using a system based on Tris–fructose egg yolk as extender to constitute semen doses of 100 × 106 spermatozoa. Post-thaw survival and acrosomal status of cells were assessed by dual staining by Hoechst 33258 and FITC-PSA. Three different forms of fluorescence distribution were displayed indicating sperm without acrosome (unstained cells), sperm with damaged acrosome (cells with incomplete fluorescence over the head), sperm with widespread fluorescence (cells completely fluorescent). Motility and kinetic rating at thawing and after 1 and 3 h incubation (37 ◦ C) were also assessed. Semen frozen in summer and autumn, corresponding to the breeding season, showed the highest (P < 0.01) post-thaw survival of spermatozoa (41.7%) and the lowest (P < 0.01) incidence of spermatozoa with damaged acrosome. The positive influence of the summer–autumn period was expressed also on motility and kinetic rating of spermatozoa at thawing. The integrity of the acrosomal membrane was positively correlated (P < 0.01) with sperm viability before processing (r = 0.32) and after thawing (r = 0.51). In conclusion, the results show that season exerts a significant influence on semen freezability in Leccese ram, with the best performance occurring the summer and autumn period, corresponding to the reproductive season in temperate zones. © 2003 Elsevier B.V. All rights reserved. Keywords: Sheep-male reproduction; Seasonal dynamics; Cryopreservation

1. Introduction It is well known that in temperate zones of the Northern Hemisphere (above 40◦ N) season influences reproductive activity of rams, affecting testicular size (D’Occhio et al., 1984; ∗ Corresponding author. Tel.: +39-080-5442825; fax: +39-080-5442828. E-mail address: [email protected] (A.G. D’Alessandro).

0378-4320/03/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-4320(03)00113-1

94

A.G. D’Alessandro, G. Martemucci / Animal Reproduction Science 79 (2003) 93–102

Martemucci et al., 2000), gonadal endocrine patterns (Lincoln et al., 1990; Martemucci et al., 2000), quantitative and qualitative sperm production (Pelletier et al., 1988; Pérez et al., 1997) and sexual behavior (Poulton and Robinson, 1987; Martemucci et al., 2000) due to the variations of photoperiod and environmental cues (Colas, 1983; Colas et al., 1986; Martin et al., 1990). Moreover, the influence of season has been reported on the fertilizing ability of spermatozoa after cryopreservation (Colas and Brice, 1976; Guérin et al., 1992), which is an important limiting factor for the large scale application of artificial insemination with frozen semen. Various extenders have been studied for the dilution of semen before freezing (review: Maxwell and Salamon, 1993; Salamon and Maxwell, 1995a, 2000) and fundamentally they can be traced back to two types, one based on Tris–egg yolk-pellet method and one on milk–egg yolk diluent in straws (Cogniè, 1990). The aim of the present study was to examine seasonal variations of freezability of ram semen following a freezing system in straws based on Tris–fructose egg yolk as extender to constitute semen doses of 100 × 106 spermatozoa, in order to establish the period more suitable to semen freezing to employ in artificial insemination.

2. Materials and methods Semen was collected from five adult Leccese rams kept in semi-intensive conditions on the farm of the Faculty of Agricultural Science, Bari University (southern Italy, 41◦ N). The rams were subjected to a rhythm of collection twice a week throughout 1 year by artificial vagina. Every 2 weeks, the quantitative and qualitative characteristics of the semen of each ram were evaluated. Ejaculates having a sperm concentration of more than 3 × 109 spermatozoa per millilitre, kinetic rating 4/5 (scale 1–5) and 60% motile cells were processed for freezing. Throughout the whole experiment, samples of the collected fresh semen were analysed for viability, motility and acrosomal integrity of spermatozoa. Then, semen was frozen according to a freezing method based on the use of Tris–fructose egg yolk as extender (Kupferschmied and Muther, modified for ram semen by Martemucci et al., 1994). In particular, a basal extender containing Tris (2.42% (w/v)), fructose (1%), citric acid (1.36%) and antibiotics (penicillin + streptomycin), with pH = 7.2–7.4 and osmolarity of 300–325 mOsm, was employed to prepare two diluents (␣ and ␤ diluent). Diluent ␣ was made of the basal extender (67.2% (v/v)), egg yolk (20%) and sterile bidistilled water (12.8%); diluent ␤ differed from the ␣ diluent in the replacement of water with the same volume of glycerol. After a pre-dilution in a 1:1 rate (semen:diluent ␣), the semen was diluted in two steps with diluent ␣ at room temperature and, following slow cooling to 4 ◦ C in 2 h, with diluent ␤. Diluents ␣ and ␤ were added respectively in 60 and 40% of the final volume needed to obtain a pre-freezing sperm concentration of 500 × 106 spermatozoa per millilitre. Diluted semen was loaded into 0.25 ml French straws (IMV, L’Aigle, France) constituting doses of 100 × 106 spermatozoa per straw. The straws were exposed to nitrogen vapours (−75 ◦ C for 7 min) before being plunged into liquid nitrogen (−196 ◦ C) for storage.To thaw, the straws were plunged into a water bath at 37 ◦ C for 30 s. For the assessment of post-thaw survival and acrosomal status of spermatozoa, semen was diluted in phosphate buffer saline (PBS) up to 50 × 106 spermatozoa per milliliter.

A.G. D’Alessandro, G. Martemucci / Animal Reproduction Science 79 (2003) 93–102

95

Survival and acrosomal integrity of spermatozoa were assessed by dual staining with Hoechst 33258 (Sigma; Milan, Italy) and fluorescein isothiocyanate labeled Pisum sativum Agglutinin (FITC-PSA, Sigma), as described by Cross et al. (1986), with some modifications. Hoechst 33258 is a fluorescent dye which stains the dead cells blue, binding to DNA, while FITC-PSA binds to glycoproteins of acrosome, and stains sperm acrosome green, with variable color intensity depending on the level of acrosome integrity. Briefly, semen was diluted in PBS up to a concentration of 50×106 spermatozoa per milliliter. A part of the sperm suspension (100 ␮l) was combined with 50 ␮l of Hoechst solution (10 ␮l/ml in PBS) and incubated in the dark at room temperature for 15 min. The sample was then washed twice in PBS to remove excess Hoechst and centrifuged at 2800 rpm for 10 min. Sperm was pelletted and re-suspended in 100 ␮l PBS. A drop of 20 ␮l of the suspension was smeared on a slide and placed in absolute ethanol (95%) for 30 min at 4 ◦ C. After incubation, the slides were washed with PBS, thoroughly flooded with FITC-PSA (10 ␮l/ml PBS) and kept in the dark for 15 min at room temperature. The slides were washed with PBS, coverslipped and observed under an epi-fluorescence microscope (Leitz, Germany) at 1000× magnification within 2 h. A total of 150 cells were counted in duplicates for each sample, using LP 430 (340–380 nm) and LP 520 (450–490 nm) filters, respectively for Hoechst and FITC-PSA staining. According to the distribution of FITC-PSA fluorescence staining, spermatozoa with acrosome breakdown were grouped into the following three classes: sperm without acrosome (cells completely unstained), sperm with damaged acrosome (cells with patchy fluorescence), sperm with widespread fluorescence (cells completely fluorescing). Sperm motility, evaluated as percentage of motile spermatozoa and kinetic rating, was assessed on frozen and fresh semen under a microscope fitted to a warm stage (37 ◦ C). For the assessment of frozen semen, the samples were transferred to PBS and motility was evaluated at 0 h (thawing) and after 1 and 3 h incubation (37 ◦ C), maintaining the semen in PBS for the incubation period. For each sample, the percentage of motile spermatozoa was determined on 40 cells of the Makler counting chamber under a phase contrast microscope (200×) fitted with a stage warmer at 37 ◦ C. The microscope was equipped with a camera, a monitor and a Polaroid freeze-frame video recorder. For each Makler counting chamber cell, all immotile spermatozoa were counted then, after immobilizing the image on the screen of the monitor by the freeze-frame video recorder, the total number of spermatozoa was counted. Motile spermatozoa were calculated as the difference between the total number of spermatozoa and the immotile spermatozoa. The kinetic rating was determined under a microscope (400×) on a drop of diluted semen (1:100 in PBS) at thawing and after 1 and 3 h incubation (37 ◦ C) according to a scale from 0 to 5 score (0: no motion; 5: very rapid forward progression). The experimental data were referred as seasonal means: winter (January to March), spring (April to June), summer (July to September) and autumn (October to December). 2.1. Statistical analyses Before statistical analysis, the data were tested for homogeneity of variance by the Bartlett’s test and, where necessary, were transformed (log+1). Data were analyzed by least squares analysis of variance using the GLM procedure of the SAS system (SAS, 1987). The statistical model used considered the effects of season, animal and their interactions. Least

96

A.G. D’Alessandro, G. Martemucci / Animal Reproduction Science 79 (2003) 93–102

squares means among seasons were compared by the t-value (PDIFF option). Coefficients of correlation between the different parameters were also calculated by regression analysis (SAS, 1987).

3. Results Seasonal variations of quantitative and qualitative characteristics of fresh semen were reported in another study (D’Alessandro et al., 2001). In this study, some results relative to fresh semen were reported in comparison with frozen semen. The percentage of live spermatozoa estimated in fresh semen as a mean of the whole year was 75%. Cryopreservation of semen significantly influenced (P < 0.01) post-thaw sperm viability reducing it in all the seasons (P < 0.01; data not shown). Post-thaw survival of spermatozoa was affected (P < 0.05) by season and individual ram effect. The survival rate of spermatozoa was highest in semen frozen in summer (P < 0.01) without difference compared to autumn, while the lowest sperm viability was shown in spring (0.05 > P < 0.01) (Table 1). The integrity of the acrosomal membrane of spermatozoa in fresh semen, estimated at 81.0% as annual mean, was significantly (P < 0.01) impaired by freezing process. The incidences of spermatozoa with acrosomal breakdown was affected by season (P < 0.01) and resulted lowest in summer and autumn (P < 0.01) and highest in spring (Table 1). Significant (P < 0.01) was the effect of individual ram. The percentage of spermatozoa having intact acrosome at thawing resulted positively correlated (P < 0.01) with the percentages of live sperm both in unprocessed semen and after thawing (r = 0.28 and 0.51, respectively). The different classes of post-thaw acrosomal injures of sperm assessed by FITC-PSA are reported in Table 2. On percentage of spermatozoa without acrosome significant (P < 0.01) were the influences of season and individual ram. Among the seasons, the maximum value was shown in spring (P < 0.01). Of the total abnormalities relative to sperm acrosome, damage of acrosome detected as patchy fluorescence of the membrane was the most frequent, and took place mainly in winter showing significant differences compared to summer (P < 0.01) and autumn (P < 0.05). Percentages of sperm without acrosome and sperm with damaged acrosome were negatively correlated (P < 0.01) during the whole year (r = −0.48). Table 1 Seasonal variations of live spermatozoa after thawing and post-thaw acrosomal break down of spermatozoa in Leccese ram (mean ± S.E.M.) Period

Live sperm (%)

Sperm with acrosome break down (%)

Winter Spring Summer Autumn

38.5 ± (38.4–38.9) 33.0 ± 1.9Bb (29.8–35.4) 42.6 ± 1.7A (33.5–50.5) 41.7 ± 2.0A (38.5–46.3)

70.5 ± 1.9B (56.8–79.0) 77.9 ± 2.3A (73.3– 86.2) 64.3 ± 2.1C (60.2–71.5) 65.2 ± 2.4BC (62.1–71.5)

1.5a

Values with different superscripts in the same column indicate differences among seasons (A–C) P < 0.01; (a–b) P < 0.05. The range of seminal characteristics for each season is shown in parentheses.

A.G. D’Alessandro, G. Martemucci / Animal Reproduction Science 79 (2003) 93–102

97

Table 2 Seasonal variations of post-thaw acrosome status of spermatozoa in Leccese ram (mean ± S.E.M.) Acrosome—break down sperm (%)

Winter Spring Summer Autumn

Loss of acrosome

Damage of acrosome

Sperm with widespread fluorescence

8.2 ± 1.3B (6.5–10.8) 19.0 ± 1.6A (12.0–23.2) 9.7 ± 1.5B (8.4–10.9) 10.9 ± 1.8B (9.3–12.7)

62.1 ± 2.0Aa (49.3–72.4) 58.4 ± 2.5 (48.8–73.2) 54.1 ± 2.3B (51.6–59.9) 53.9 ± 2.6b (48.9–62.2)

0.2 ± 0.1 (0.1–0.3) 0.5 ± 0.1 (0–1.3) 0.4 ± 0.1 (0.2–0.7) 0.4 ± 0.1 (0–0.6)

Values with different superscripts in the same column indicate differences among seasons (A–C) P < 0.01; (a–b) P < 0.05. The range of seminal characteristics for each season is shown in parentheses.

Table 3 Seasonal variations of percentage of motile spermatozoa at thawing (0 h) and after 1 and 3 h incubation (37 ◦ C) in Leccese ram (mean ± S.E.M.) Motile spermatozoa (%) after incubation 0h Winter Spring Summer Autumn

19.6 ± (13.9–24.4) 27.3 ± 2.7 (22.0–34.6) 26.3 ± 2.4 (21.2–32.0) 29.4 ± 2.9a (26.0–32.5) 3.5b

1h

3h

4.7 ± 3.0 8.8 ± 2.3 9.3 ± 2.0 8.7 ± 2.4

1.0 ± 1.7 (0.2–1.9) 1.8 ± 1.3 (0.8–2.9) 1.5 ± 1.1 (0.1–3.3) 1.9 ± 1.3 (1.0–3.0)

Values with different superscript on the same column indicate differences among seasons (a–b) P < 0.05. The range of seminal characteristics for each season is shown in parentheses.

The percentage of cells showing widespread FITC-PSA fluorescence was low (<1.0%). The percentage of motile sperm, a mean of 64.4% in fresh semen, was significantly impaired by the freezing process (P < 0.01). Sperm motility decreased drastically (P < 0.01) after 1 h from thawing and even more after 3 h incubation, when it reached the value of 1.0–1.9% (P < 0.01) (Table 3). The percentage of motile sperm after 3 h incubation was negatively correlated with sperm motility at thawing (r = −0.59; P < 0.01), while no correlations were found considering motility of fresh semen. Table 4 Seasonal variations of kinetic rating (scale 0–5) of spermatozoa at thawing (0 h) and 1 and 3 h incubation (37 ◦ C) in Leccese ram (mean ± S.E.M.) Kinetic rating after incubation 0h Winter Spring Summer Autumn

1h

3.24 ± (2.5–4.0) 3.63 ± 0.10a (3.5–3.8) 3.68 ± 0.09A (3.3–4.0) 3.78 ± 0.11A (3.5–4.0) 0.13Bb

3h

2.1 ± 2.7 ± 0.2a 2.9 ± 0.1A 2.5 ± 0.2

0.2Bb

0.44 ± 0.24 (0.2–0.7) 0.75 ± 0.18 (0.6–0.8) 0.55 ± 0.16 (0.2–1.3) 0.42 ± 0.19 (0–0.8)

Values with different superscripts in the same column indicate differences among seasons (A–C) P < 0.01; (a–b) P < 0.05. The range of seminal characteristics for each season is shown in parentheses.

98

A.G. D’Alessandro, G. Martemucci / Animal Reproduction Science 79 (2003) 93–102

The kinetic rating of spermatozoa followed the same trend of motility. At the third hour of incubation, the kinetic rating (on a scale from 0 to 5) decreased drastically (P < 0.01) to 1.0 (range 0.42–0.75) without any differences among the seasons (Table 4).

4. Discussion The effects of cryopreservation of semen at low temperature are well documented (Parks and Graham, 1992; Watson, 1995, 1996, 2000; Salamon and Maxwell, 1995b; Maxwell and Watson, 1996). In previous studies on the same Leccese breed ram, we found that sperm concentration was not influenced by the season while the best qualitative characteristics of spermatozoa correspond to the decreasing photoperiod of summer and autumn (D’Alessandro et al., 2001). The total number of spermatozoa per ejaculate was highest in spring, without significant difference when compared to the autumn (D’Alessandro et al., 2001). Testicular endocrine function was at maximum level in spring and summer, with the peak occurring in July, exactly 1 month before the best performance in terms of testicular size and libido (Martemucci et al., 2000). In this study, we wanted to evaluate the effects of season on freezability of semen in Leccese ram, a breed with a certain sensitivity to season variations, in order to establish the most suitable period for freezing semen with low sperm number in straw for use in artificial insemination. There are reports on seasonal differences between breeds in concerning endocrine pituitary and gonadal activities (D’Occhio et al., 1984; Boland et al., 1985), sperm concentration (Dufour et al., 1984; Bonanno et al., 1990; Mandiki et al., 1998) and also freezability of spermatozoa (Maxwell, 1980; Cournock et al., 1984). Considering the pattern shown by the fresh semen (D’Alessandro et al., 2001), it is possible to verify that cold spermatozoa resistance after freezing was affected by the quality of semen before processing. In fact, the highest values of post-thaw viability were recorded during the second semester of the year (summer and autumn), when the fresh semen shows the highest percentage of live cells (81.0%; D’Alessandro et al., 2001). During the spring, post-thaw acrosomal breakdown was highest. Resistance of the acrosomal membrane is strictly dependent on plasma membrane integrity, since percentages of spermatozoa with intact acrosome are positively correlated to post-thaw viability. The effect of the freezing process on sperm membrane integrity in relation to the season may be attributable to the seasonal variations in the composition of ram seminal plasma and protein contents, which show higher levels during the breeding season compared with the non-breeding season (Smith et al., 1999). It was supposed that seasonal changes in gonadothophin levels (Xu et al., 1991) and their receptors in the testis (Barenton and Pelletier, 1983) affect endocrine gonad function and secretions of seminal vesicles and epididymis (Smith et al., 1999). It is known that cooling or cryopreservation of sperm induces a destabilization of the membranes leading to a status similar to capacitation and acrosome reaction (Watson, 1995, 1996; Maxwell and Watson, 1996) and spermatozoa viability is correlated with acrosomal integrity (Valcárcel et al., 1997). In the present study the freezing process determines a high percentage of spermatozoa with patchy FITC-PSA fluorescence, an index of acrosome reaction in progress (Sukardi et al., 1997). Moreover, a negative correlation was shown between percentages of sperm without acrosome and sperm with damaged acrosome during the

A.G. D’Alessandro, G. Martemucci / Animal Reproduction Science 79 (2003) 93–102

99

whole year, demonstrating differing sensitivity of spermatozoa within the whole population to the freezing system. This finding could be an expression of the phenomenon of heterogeneity, the functional variability in the response of spermatozoa subjected to the same conditions (Watson, 1996). Motility of spermatozoa at thawing is considered a valid test to evaluate the quality of frozen semen from the point of view of cells functionality (Leboeuf, 1989). It is well known that freezing and thawing impairs cells structures and function reducing motility as well as fertilizing capacity of sperm (Watson, 1995, 1996). The reduction in motility has been attributed to several events which concern changes in the active transport and permeability of the plasma membrane in the tail region, damage to the axonemal elements, loss of internal mitochondrial structure, and alteration of energy (ATP) production (Sherman and Liu, 1982; Courtens et al., 1989; Watson, 1996). Moreover, the reduction of total protein concentration and absence of specific protein from seminal plasma, due to seasonal influence, was associated to the lower sperm motility in frozen semen (Smith et al., 1999). On the basis of these findings, it could be interesting to gain further knowledge of seasonal effect on semen freezability by investigating the role of seminal plasma. An indirect test to determine the effect of seminal plasma variations on cryosurvival of spermatozoa would be represented by mixing some seminal plasma frozen in spring with some seminal spermatozoa frozen in autumn, and vice versa. Seminal plasma is believed to contain decapacitation factors (Oliphant and Brackett, 1973) that could contrast the capacitation-like changes of spermatozoa induced by freezing/thawing processes (Gillan and Maxwell, 1999). It was demonstrated that the addition of seminal plasma to cryopreserved spermatozoa resulted in normal fertility after cervical artificial insemination (Maxwell et al., 1999). Moreover, it would be interesting to evaluate fertilising ability of semen frozen in different seasons. Semen frozen in autumn showed higher freezability and fertility than semen processed in spring (Ile de France and Berrichonne du Cher, latitude 47◦ N: Colas and Brice, 1976; Guérin et al., 1992), while in other breeds no seasonal (spring versus autumn) difference was observed in the lambing rate (Suffolk, Texel rams breeds, latitude 54◦ N: Maxwell, 1980; Cournock et al., 1984). Sperm motility after 3 h incubation was markedly reduced and resulted correlated negatively with motility after thawing. This could be attributable to the shortened life of the cells which might be associated with acrosomal-like reaction induced by freezing process (Watson, 1995, 1996; Maxwell and Watson, 1996). This suggestion would be supported by the positive relationship between post-thaw acrosome integrity and motility of spermatozoa found in ram (Salamon and Maxwell, 1995b) and in buck (Martemucci et al., 1999) semen. In this study, a marked variations between rams were shown on spermatozoa freezability, confirming the results reported by other researchers (Maxwell, 1986; Butler and Maxwell, 1988; Eppleston and Maxwell, 1991). Differences between rams have been attributed to both genetic and environmental conditions (Salamon and Maxwell, 1995b). In conclusion, we have shown that freezability of Leccese ram semen was better during the summer and autumn seasons. In particular, autumn represents the best season both for semen collection and for freezing, because during this period a higher number of spermatozoa per ejaculate (D’Alessandro et al., 2001) as well the best freezability characteristics of spermatozoa were observed.

100

A.G. D’Alessandro, G. Martemucci / Animal Reproduction Science 79 (2003) 93–102

Acknowledgements This research was supported by MURST (ex 60%). The authors wish to thank L. Bongermino for statistical processing analysis and L. Basso, P. Cataldo and D. D’Elia for technical assistance in conduction of the experiment.

References Barenton, B., Pelletier, J., 1983. Seasonal changes in testicular gonadotrophin receptors and steroid content in the ram. J. Endocrinol. 112, 1441–1446. Boland, M.P., Al-kamali, A.A., Crosby, T.F., Haynes, N.B., Howles, C.M., Kelleher, D.L., Gordon, I., 1985. The influence of breed, season and photoperiod on semen characteristics, testicular size, libido and plasma hormone concentrations in rams. Anim. Reprod. Sci. 9, 241–252. Bonanno, A., Alabiso, M., Portolano, B., Alicata, M.L., 1990. Valutazione spermatica e prove di inseminazione artificiale su ovini Comisani in Sicilia (Spermatic evaluation and trials of artificial insemination in Comisana sheep breed in Sicilia). In: Proceedings of Giornate Siculo-Tunisine su Zootecnia e Zoonosi. Palermo, Italy, pp. 119–136. Butler, L.G., Maxwell, W.M.C., 1988. The effects of sire and location on results of on-farm intrauterine insemination of frozen-thawed semen. Proc. Aust. Assoc. Anim. Breed. Genet. 7, 394–397. Cogniè, Y., 1990. Current technologies for synchronization and artificial insemination in sheep. In: Oldham, C.M., Martin, G.B., Purvis, I.W. (Eds.), Reproductive Physiology of Merino Sheep. The University of Western Australia, Perth, pp. 207–216. Colas, G., 1983. Factors affecting the quality of ram semen. In: Haresign, W. (Ed.), Sheep Production. Butterworks, London, UK, pp. 453–465. Colas, G., Brice, G., 1976. Seasonal variations of the fertilizing capacity of deep-frozen ram semen. In: Proceedings of the Eighth International Congress on Animal Reproduction and Artificial Insemination, vol. 4. Crakow, Poland, pp. 977–980. Colas, G., Guerin, Y., Lemaire, Y., Montassier, Y., Despierres, J., 1986. Seasonal variations in testicular diameter and spermatozoa morphology of Vendean and Texel rams. Reprod. Nutr. Dev. 26, 863–875. Cournock, R.M., Reed, H.C.B., Logue, D.N., Maxwell, W.M.C., 1984. Artificial insemination of ewes with ram semen frozen by the pellet method. Anim. Prod. 38, 546 (abstr.). Courtens, J.L., Ekwall, H., Paquignon, M., Ploen, L., 1989. Preliminary study of water and some element contents in boar spermatozoa before, during and after freezing. J. Reprod. Fertil. 87, 613–626. Cross, N.L., Morales, P., Overstreet, J.W., Hanson, F.W., 1986. Two simple methods for detecting acrosome reacted human sperm. Gamete Res. 15, 213–226. D’Alessandro, A.G., Colonna, M.A., Bellitti, A., Martemucci, G., 2001. Variazioni durante l’anno delle caratteristiche quantitative e qualitative del seme in arieti di razza Leccese (Variations during the year in quantitative and qualitative semen characteristics in Leccese rams). Zoot. Nutr. Anim. 27, 221–230. D’Alessandro, A.G., Martemucci, G., Colonna, M.A., Bellitti, A., 2001. Post-thaw survival of ram spermatozoa and fertility after insemination as affected by pre-freezing sperm concentration and extender composition. Theriogenology 55, 1159–1170. D’Occhio, M.J., Kinder, J.E., Schanbacker, B.D., 1984. Profiles of luteinizing hormone, follicle stimulating hormone, testosterone and prolactin in rams of diverse breeds: effect of contrasting short (8L:16D) and long (16L:8D) photoperiods. Biol. Reprod. 30, 1039–1054. Dufour, J.J., Fahmy, M.H., Minvielle, F., 1984. Seasonal changes in breeding activity, testicular size, testosterone concentration and seminal characteristics in rams with long or short breeding season. J. Anim. Sci. 58, 416–422. Eppleston, J., Maxwell, W.M.C., 1991. Between ram variation in the reproductive performance of ewes inseminated with frozen semen. Proc. Aust. Soc. Reprod. Biol. 23, 130 (abstr.). Gillan, L., Maxwell, W.M.C., 1999. The functional integrity and fate of cryopreserved ram spermatozoa in the female tract. J. Reprod. Fertil. Suppl. 54, 271–283.

A.G. D’Alessandro, G. Martemucci / Animal Reproduction Science 79 (2003) 93–102

101

Guérin, Y., Cognié, Y., Poulin, N., 1992. Freezability of freshly ejaculated and frozen ram semen in vitro. In: Proceedings of the 12th International Congress on Animal Reproduction, vol. 3. The Hague, The Netherlands, pp. 1418–1420. Leboeuf, B., 1989. L’insémination artificielle caprine en France, état actuel et perspectives d’avenir. In: Enne, G., Greppi, G.F. (Eds.), Atti Simp. Int. La riproduzione dei piccoli ruminanti: basi fisiologiche ed aspetti applicativi. CCIA Varese Ist. Sper. It. “L Spallanzani” Milano, APA Varese, Italy, pp. 87–113. Lincoln, G.A., Lincoln, C.E., McNeilly, A.S., 1990. Seasonal cycles in the blood plasma concentration of FSH, inhibin and testosterone, and testicular size in ram of wild, feral and domesticated breeds of sheep. J. Reprod. Fert. 88, 623–633. Mandiki, S.N.M., Derycke, G., Bister, J.L., Paquay, R., 1998. Influence of season and age on sexual maturation on parameters of Texel, Suffolk and Ile-de-France rams. I. Testicular size, semen quality and reproductive capacity. Small Rum. Res. 28, 67–79. Martemucci, G., Bramante, G., D’Alessandro, A., Gambacorta, M., Facciolongo, A.M., Iaffaldano, N., 1994. Valutazione della inseminazione intrauterina con seme congelato: effetto della dose sul tasso di concepimento. Risultati preliminari (Intrauterine insemination in sheep with frozen semen: effects of dose on conception rates. Preliminary results). In: Proceedings of the VI Meeting on Studio dell’efficienza riproduttiva degli animali di interesse zootecnico. Bergamo, Italy, pp. 87–91. Martemucci, G., D’Alessandro, A.G., Laera, A., Iaffaldano, N., 1999. Congelabilità del seme in becchi di razza Maltese:effetti delle variazioni stagionali e del tasso di diluizione sullo status dell’acrosoma e sulla vitalità dei nemaspermi (Freezability of Maltese buck semen: effects of seasonal variations and pre-freezing dilution rate on acrosomal status and survival of frozen-thawed spermatozoa). Zoot. Nutr. Anim. 25, 91–103. Martemucci, G., Facciolongo, A.M., Bramante, G., 2000. Variazioni durante l’anno delle dimensioni testicolari, della libido e della testosteronemia successiva a stimolazione con Gn-RH in arieti di razza Leccese (Circannual variations in testis size, libido and testosterone hormone response to Gn-RH in Leccese rams). Zoot. Nutr. Anim. 26, 199–209. Martin, G.B., Ford, J.R., Purvis, I.W., 1990. Environmental and genetics factors affecting reproductive activity in the Merino ram. In: Oldham, C.M., Martin, G.B., Purvis, I.W. (Eds.), Reproductive Physiology of Merino Sheep: Concepts and Consequences. School of Agriculture (Animal Science), The University of Western Australia, Perth, pp. 111–129. Maxwell, W.M.C., 1980. Fertility of ram semen frozen in autumn and spring. Proc. Aust. Soc. Reprod. Biol. 12, 8 (abstr.). Maxwell, W.M.C., 1986. Artificial insemination of ewes with frozen-thawed semen at a synchronized oestrus. 2. Effect of dose of spermatozoa and site of insemination on fertility. Anim. Prod. Sci. 10, 301–308. Maxwell, W.M.C., Salamon, S., 1993. Liquid storage of ram semen: a review. Reprod. Fertil. Dev. 5, 613–638. Maxwell, W.M.C., Watson, P.F., 1996. Recent progress in the preservation of ram semen. Anim. Reprod. Sci. 42, 55–65. Maxwell, W.M.C., Evans, G., Mortimer, S.T., Gillan, L., Gellatly, E.S., McPhie, C.A., 1999. Normal fertility in ewes after cervical insemination with frozen-thawed spermatozoa supplemented with seminal plasma. Reprod. Fertil. Dev. 11, 123–126. Oliphant, G., Brackett, B.G., 1973. Immunological assessment of surface changes of rabbit sperm undergoing capacitation. Biol. Reprod. 9, 404–414. Parks, J.E., Graham, J.K., 1992. Effects of cryopreservation procedures on sperm membranes. Theriogenology 38, 209–222. Pelletier, J., Chemineau, P., Delgadillo, J.A., 1988. Seasonality of sexual activity and its photoperiodic control in the adult ram and he-goat. In: Proceedings of the 11th International Congress on Animal Reproduction and Artificial Insemination. Dublin, Ireland, pp. 211–219. Pérez, R., Lòpez, A., Castrillejo, A., Bielli, A., Laborde, D., Gastel, T., Tagle, R., Queirolo, D., Franco, J., Forsberg, M., Rodr`ıguez-Mart`ınez, H., 1997. Reproductive seasonality of Corredale rams under extensive rearing conditions. Acta Vet. Scand. 38, 109–117. Poulton, A.L., Robinson, T.J., 1987. The response of rams and ewes of three breeds to artificial photoperiod. J. Reprod. Fertil. 79, 609–626. Salamon, S., Maxwell, W.M.C., 1995a. Frozen storage of ram semen. I. Processing, freezing, thawing and fertility after cervical insemination. Anim. Reprod. Sci. 37, 185–249.

102

A.G. D’Alessandro, G. Martemucci / Animal Reproduction Science 79 (2003) 93–102

Salamon, S., Maxwell, W.M.C., 1995b. Frozen storage of ram semen. II. Causes of low fertility after cervical insemination and methods of improvement. Anim. Reprod. Sci. 46, 89–96. Salamon, S., Maxwell, W.M.C., 2000. Storage of ram semen. Anim. Reprod. Sci. 62, 77–111. SAS 1987, SAS/STAT TM Guide for Personal Computers, Version 6. SAS Institute Inc., Cary, NC, 1029 pp. Sherman, J.K., Liu, K.C., 1982. Ultrastructure before freezing, while frozen, and after thawing in assessing cryoinjury of mouse epididimal spermatozoa. Cryobiology 19, 503–510. Smith, J.F., Parr, J., Murray, G.R., McDonald, R.M., Lee, R.S.-F., 1999. Seasonal changes in the protein content and composition of ram seminal plasma. In: Proceedings of the New Zealand Society of Animal Production, vol. 59. pp. 223–225. Sukardi, S., Curry, M.R., Watson, P.F., 1997. Simultaneous detection of the acrosomal status and viability of incubated ram spermatozoa using fluorescent markers. Anim. Reprod. Sci. 46, 89–96. Valcárcel, A., De Las Heras, M.A., Perez, L., Moses, D.F., Baldassarre, H., 1997. Assessment of the acrosomal status of membrane-intact ram spermatozoa after freezing and thawing, by simultaneous lectin/Hoechst 33258 staining. Anim. Reprod. Sci. 45, 299–309. Watson, P.F., 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 7, 871–891. Watson, P.F., 1996. Cooling of spermatozoa and fertilizing capacity. Reprod. Domest. Anim. 31, 135–140. Watson, P.F., 2000. The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 60-61, 481–492. Xu, Z.Z., McDonald, M.F., McCutcheon, S.N., Blair, H.T., 1991. Seasonal variation in testis size, gonadotrophin secretion and pituitary responsiveness to GnRH in rams of two breeds differing in time of onset of the breeding season. Anim. Reprod. Sci. 26, 281–292.