104 J. Roca et al. Animal Reproduction Science 64 2000 103–112
viability and fertilizing capability of rabbit spermatozoa stored at 15
◦
C. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Rabbit; Spermatozoa; Tris extender; Cool storage; Artificial insemination
1. Introduction
Artificial insemination AI has been practised in the rabbit meat industry for over 15 years Sinkovicks et al., 1983. In order to improve breeding management, AI is used
routinely in many of the large rabbit farms in a number of European countries including Italy, France, Spain and Hungary. For practical application, rabbit AI is usually carried out
with fresh diluted semen within 6–12 h of collection, resulting in conception rates as high as those obtained with natural mating.
Despite the use of AI in the large rabbit farms of several European countries, rabbit AI has certainly not become a common practice in the rabbit meat producing areas of the
world. A limiting factor for a more extensive commercial application is related to the semen preservation. The current practice of using freshly diluted semen is mostly limited to AI of
does on the farm where the buck is located and within a few hours of semen collection. This short life places considerable constraints on rabbit AI since rapid delivery is essential. Since
frozen semen is not currently suitable for routine AI in market rabbit production for a review see Morrell, 1995 and Castellini, 1996, chilled and stored semen for 2–3 days may facilitate
the semen transport and subsequent widespread use of artificial insemination in rabbits.
The aim of the present study was to evaluate the viability of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15
◦
C and to determine its conception and farrowing rates in a commercial breeding program in order to improve the appeal of using AI among
meat producing rabbit industry.
2. Materials and methods
2.1. Animals All males and females were sexually mature hybrid rabbits. They were housed in flat
deck cages in rooms with controlled light 16:8 h L:N. Room temperature ranged from 16 to 28
◦
C. Animals received a commercial diet Purina® according to their reproductive condition. Water was provided ad libitum.
2.2. General procedure 2.2.1. Semen collection, processing and assessment of spermatozoa viability
Semen was collected four times a week with an artificial vagina from male rabbits housed at a commercial AI center CUNISANT, Murcia, Spain. After semen collection, any gel
plug was removed. Only ejaculates with good wave motion ≥3 on a 0–5 scale were used.
J. Roca et al. Animal Reproduction Science 64 2000 103–112 105
The concentration of spermatozoa was ≥ 300 × 10
6
cells ml
− 1
and the proportion of motile cells was ≥75.
The assessment of spermatozoa viability included the percentage of total sperm motility TSM, forward progressive motility FPM, plasma membrane integrity PMI and acro-
some integrity normal apical ridge, NAR. To evaluate the TSM percentage and FPM score, three samples of the diluted spermatozoa after 10 min incubation at 37
◦
C were placed under a coverslip in the center of a pre-warmed 39
◦
C slide and transferred to a heated microscope stage set at 39
◦
C and subjectively assessed by phase contrast microscopy ×100 and ×200 magnifications. The TSM was estimated in ranges of 5 and the proportion of spermato-
zoa with FPM was determined using an arbitrary scale of 0–5 0, 1, 2, 3, 4 or 5 = 0–10, 10–25, 25–50, 50–70, 70–90, or 90–100, respectively, of the motile spermatozoa showing
progressive movement. The percentage of spermatozoa with PMI was evaluated using a flu- orescence microscope magnification ×400 using a standard fluorescein filter set Nikkon,
Japan after staining of spermatozoa with 6-carboxyfluorescein diacetate Sigma Chemical, St. Louis, MO, USA as described previously by Harrison and Vickers 1990. Percentage of
spermatozoa with NAR was assessed by viewing wet mounts of diluted spermatozoa fixed in buffered 2 glutaraldehyde solution Pursel and Johnson, 1974. NAR was examined
by phase-contrast microscopy using a ×100 oil immersion objective. For PMI and NAR evaluation, a total of 200 spermatozoa were counted.
2.2.2. Synchronization of oestrous, induction of ovulation, insemination and pregnancy diagnosis
Female rabbits housed in a commercial farm were induced to oestrous synchronously with subcutaneous application of PMSG 20 UI, Folligon®, Intervet, Holland 52–56 h before AI.
To induce ovulation, does were injected i.m. with 0.8 mg of gonadotropin-releasing hormone analogue Buserelin, Suprefact®, Hoechst-Roussel, Germany at the time of insemination.
Inseminations were performed at one time in a fixed day of the week with a dose of 0.5 ml of diluted semen containing 15 × 10
6
spermatozoa. The insemination procedure was similar to that described by Boussin 1989.
AI was performed in nulliparous, primiparous and multiparous does. Nulliparous does were inseminated at 17–22 weeks of age, primiparous and multiparous does were insemi-
nated 11 days after parturition. Pregnancy diagnosis conception rate was determined by abdominal palpation on day 14 after AI. Non-pregnant does were inseminated again 21
days after the first non-fertile insemination re-breed does. The farrowing rates and litter size were recorded at the time of farrowing.
2.3. Experimental design 2.3.1. Experiment 1
The objective of the first experiment was to evaluate the viability of rabbit spermatozoa diluted in Tris-based extenders and stored at 15
◦
C for 96 h. Semen was collected from 12 male rabbits and ejaculates with adequate motility and
sperm concentration were pooled and divided into four aliquots. Each one of the four aliquots was extended to 30–40 × 10
6
spermatozoa ml
− 1
1:10, semenextender in one of the fol- lowing extenders: Tris-citric-glucose TCG, Tris-citric-fructose TCF, TES-Tris-glucose
106 J. Roca et al. Animal Reproduction Science 64 2000 103–112
Table 1 Chemical composition of Tris-buffer extenders used to dilute and preserve rabbit semen at 15
◦
C Components mM
Extender
a
TCG TCF
TTG TTF
Tris
b
313.79 313.79
56.15 56.15
TES
c
– –
238.09 238.09
Citric acid 103.07
103.07 –
– Glucose
33.3 –
33.3 –
Fructose –
33.3 –
33.3 Kanamycin mgl
80 80
80 80
pH 6.9
6.9 6.9
6.9 Osmotic pressure mOsml
336 333
348 344
a
TCG: Tris-citric-glucose, TCF: Tris-citric-fructose, TTG: TES-Tris-glucose and TTF: TES-Tris-fructose.
b
Tris: Trishydroxymethylaminomethane.
c
TES: N-trishydroxymethylmethyl-2-aminoethanesulfonic acid.
TTG and TES-Tris-fructose TTF Table 1. The extended semen samples were cooled slowly and held at 15
◦
C for 96 h. Assessment of spermatozoa viability was determined 0, 24, 48, 72 and 96 h after the beginning of cooling. This experimental procedure was repeated
six times. 2.3.2. Experiment 2
The objective of the second experiment was to determine the fertility rates of spermatozoa diluted in a Tris-based extender and stored at 15
◦
C between 0 and 48 h. Results of the Experiment 1 led us to choose the TCG extender to use in this fertility trial because it
maintained adequate sperm viability over 48 h, and was commercially affordable. Semen from 30 rabbits was used in this experiment. After collection and evaluation of
semen quality, pooled gel-free ejaculates were diluted to 30×10
6
spermatozoa ml
− 1
in TCG extender. AI was performed once using either fresh diluted semen n: 1275 and diluted
semen stored at 15
◦
C for 24 h n: 1503 or 48 h n: 935. Inseminations were performed throughout the 12 months and fertility was measured by conception rate percentage of
pregnant rabbits after palpation, farrowing rate and litter size.
2.4. Statistical analysis Calculations were made using the SPSS 9.0PC statistics package SPSS Inc., Chicago,
IL, US. Data from Experiment 1, after arcsin transformation of the percentages, were examined by ANOVA using a repeated-measures general linear model where storage time
was the repeated measure. Means ± S.E.M. for sperm parameters were compared using Duncan’s multiple range test. In Experiment 2, data from both conception and farrowing
rates the data were modeled according to the binomial model of parameters, as described by Fisz, 1980 and litter size were analyzed by two-way analysis of variance using a general
linear model. When ANOVA revealed a significant effect, values were compared using Tukey-test. Differences were considered to be statistically different at P 0.05.
J. Roca et al. Animal Reproduction Science 64 2000 103–112 107
3. Results
