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Successful artificial insemination using semen frozen and stored by an ultrafreezer in the Majorera goat breed
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Theriogenology 71 (2009) 1307–1315 www.theriojournal.com
Successful artificial insemination using semen frozen and stored by an ultrafreezer in the Majorera goat breed M. Batista a,*, T. Nin˜o a, D. Alamo a, N. Castro b, M. Santana a, F. Gonza´lez a, F. Cabrera a, A. Gracia a a
Obstetrics and Reproduction, Faculty of Veterinary of Las Palmas, Transmontan˜a s/n, 35413 Arucas, Spain b Animal Science, Faculty of Veterinary of Las Palmas, Transmontan˜a s/n, 35413 Arucas, Spain Received 27 October 2008; received in revised form 18 December 2008; accepted 29 December 2008
Abstract The semen of five Majorera breed bucks was collected and processed to reach a final concentration of 200 106 spermatozoa/ straw in the extender containing 4% of glycerol and 12% of egg yolk. Two freezing techniques were assessed: (LN) straws were frozen and stored in liquid nitrogen, and (ULF) straws were frozen and stored in the ultra-low freezer at 152 8C. Semen quality (sperm motility, acrosome integrity and abnormal sperm cells percentages) was determined for different storage times (1, 30, 90 and 365 days of cryopreservation). Thereafter, 150 Majorera goats were assigned to four experimental groups: for groups LN-1 (n = 40) and LN-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in liquid nitrogen, respectively, while for groups ULF-1 (n = 40) and ULF-6 (n = 35), the goats were transcervically inseminated with frozenthawed semen stored for 1 and 6 months in an ultra-low freezer at 152 8C, respectively. The pregnancy rate was determined by transabdominal ultrasound scanning; in addition, the kidding rate and prolificacy were recorded at parturition. In vitro results showed that the freezing protocol did not affect sperm quality with similar values for up to 1 year of cryopreservation. The kidding rates were not significantly different between experimental groups (43.6%, 38.5%, 42.8% and 40.0% for groups LN-1, ULF-1, LN-6 and ULF-6, respectively). In all experimental groups, the kidding rate and prolificacy were significantly higher ( p < 0.01) in multiparous than in nulliparous goats. Therefore, the in vitro results and fertility trials confirmed the efficiency of the ULF technique for freezing and storage of goat semen. # 2009 Elsevier Inc. All rights reserved. Keywords: Goat; Semen; Ultrafreezer; Fertility; Long-term storage
1. Introduction Artificial insemination (AI) with fresh, chilled or frozen-thawed semen is a basic tool in goat breeding, allowing the diffusion of caprine semen with high * Corresponding author at: Unidad de Reproduccio´n y Obstetricia, Facultad de Veterinaria de Las Palmas, Transmontan˜a s/n, 35413 Arucas, Las Palmas, Spain. Tel.: +34 92 8454356; fax: +34 92 8454301. E-mail address: [email protected] (M. Batista). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.12.024
genetic value [1,2]. In recent years, artificial insemination with fresh semen has become a common technique in goats [3–5]; however, the commercial use of frozenthawed semen has been relatively limited in caprine reproduction. As in other domestic animals, the freezing process reduces the viability of the caprine spermatozoa; kidding rate after AI with frozen-thawed semen ranged between 30% and 70% [4,6–12], while with fresh or chilled semen produces values between 60% and 80% [3,5,6,13]. With transcervical insemination, a
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low proportion of spermatozoa traverse the cervical canal, meaning a relatively higher number of sperm cells are necessary to get reasonable pregnancy rates [3,10,14]. Different studies have noted this fact as a handicap in artificial insemination in small ruminants [14,15]. Laparoscopic intrauterine insemination of goats resulted in improved fertility rates [7,16]; however, the high costs associated with this technique and the necessary qualified inseminators are restrictive factors for the use of laparoscopy to inseminate goats. The cryopreservation of semen straws in liquid nitrogen (LN) is the standard protocol to freeze and store caprine semen. Other studies have described the freezing of semen by placing pellets in dry ice at 79 8C, but these require the subsequent immersion of the pellets in LN for the frozen storage of semen [1]. In some areas, LN is not always readily available and its cost is relatively high. The development of freezedrying and vitrification methods, avoiding the use of LN, has been postulated [17] as a feasible alternative for sperm cryopreservation. However, in vivo trials have not been developed to confirm the effectiveness of these alternative strategies. A recent study [14] in goats did report high fertility rates after artificial insemination with semen frozen using a directional freezing machine; however, this study also used LN for the storage of semen. In canine, different studies [18,19] have confirmed that the use of ultrafreezers at 152 8C for freezing and storing canine semen could represent a potential alternative to liquid nitrogen. Our preliminary study [20] developed in Majorera bucks assessed, for the first time, the use of ultrafreezers at 152 8C for freezing and storing caprine semen. This study showed that the in vitro seminal quality (2 months after freezing) was not significantly different for seminal straws frozen and stored in liquid nitrogen when compared with those frozen and stored exclusively in the ultra-low temperature freezer at 152 8C. However, the results of longterm storage of semen frozen using ultrafreezers at 152 8C have not been related; in addition, fertility trials to test the fertilizing capacity of semen frozen by an ultrafreezer at 152 8C has not been developed until now. The aim of this experiment was to assess the use of an ultra-low freezer at 152 8C both for freezing and for short-term (1, 3 months) and long-term (12 months) storage of caprine semen. In addition, in vivo trials were carried out to confirm the caprine semen remained fertile after cryopreservation with the ultra-low temperature freezer at 152 8C.
2. Materials and methods 2.1. Experiment 1 2.1.1. Animals Five healthy bucks of the Majorera breed, aged between 3.5 and 5 years, were selected by their breeding value. The study was conducted at the experimental farm of the Veterinary Faculty of Las Palmas (Canary Islands, Spain, 288N, 238W). The animals were housed in a collective pen, and fed was a mix of maize and bran (400 g per male) and dehydrated alfalfa; mineral salts and water were provided ‘‘ad libitum’’. All experimental work was carried out according the Spanish laws for animal research and experimentation. 2.1.2. Semen collection and evaluation During the breeding season (September–November 2006), semen was collected from each male, at 2 per week for 5 weeks, using an artificial vagina. Immediately after collection, the ejaculates were placed in a water bath (37 8C) and aliquots were taken for the assessment of semen quality. The sperm concentration, sperm motility, and the percentages of sperm cells with intact acrosome membrane integrity and with abnormal morphology were defined. The semen volume was recorded directly in a calibrated tube. The sperm concentration was determined with a Spermacue1 spectrophotometer (Minitu¨b GmbH, Germany), which had been calibrated for buck semen. Before analysis, 25 mL of each semen sample was added to 5 mL plastic tubes containing 975 mL of PBS at 37 8C, and, this solution was then, maintained at 37 8C until assessment. The proportion of motile sperm cells was evaluated subjectively using a phase contrast microscope (magnification: 1000) and with a Computer Assisted Sperm Analysis (CASA) system (Sperm Vision Lite1, Minitu¨b Ibe´rica, Tarragona, Spain); the semen variables included in the analysis were the percentage of total motile spermatozoa, percentage of progressive fast spermatozoa, the percentage of locally motile spermatozoa and the percentage of non-motile sperm cells. For each diluted semen sample, two aliquots of 10 mL were placed on a Makler chamber at 37 8C and the sperm contained in seven randomized fields in each of the two aliquots were assessed for motility; at least, 2000 sperm cells/aliquot were evaluated to define the motility parameters. The settings of the motility analyzer were as follows: number of frames, 16; frames per second, 25; velocity limit for non-motile sperm cells, 10 mm/s; velocity limit for locally motile
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sperm cells, 20 mm/s; velocity limit for low motile sperm cells, 30 mm/s; velocity limit for medium motile sperm cells, 60 mm/s; filter for particles, 20%. The acrosome integrity was assessed using Spermac staining technique (Minitu¨b, Tiefenbach, Germany) counting a minimum of 200 sperm cells per slide (phase contrast microscopy, at 1000). The percentage of cells with abnormal morphology was estimated by placing 50 mL of the semen sample on a pre-warmed (37 8C) slide; after staining with the Blom’s eosinnigrosin stain [21], the samples were assessed under a phase contrast microscopy (100) by examining 200 cells per sample. Only semen samples with progressive sperm motility >75%, acrosome intact spermatozoa >80% and normal sperm morphology >85% were used. 2.1.3. Semen processing Between 5 and 10 min after collection, the ejaculates from each male were washed to remove the seminal plasma. The ejaculate was diluted in washing solution (250 mM Tris, 28 mM glucose, 104 mM citric acid, 0.05% streptomycin and 500 UI penicillin/mL) at 37 8C, and centrifuged twice at 700 g for 15 min (room temperature, 20 8C) and the supernatant was removed. The ejaculates of each buck were processed individually. Semen processing was performed as described by Cabrera et al. [22]. Briefly, the sperm pellet was diluted (1:3) in a Tris-based freezing medium (250 mM Tris, 28 mM glucose, 104 mM citric acid, 12% egg yolk, 0.05% streptomycin, 500 UI penicillin/mL, and distilled water to 100 mL). Thereafter, the sperm concentration was determined and the volume was adjusted (at room temperature, 20 8C) to reach a final concentration of 800 106 spermatozoa/mL. After 5 min, a new dilution was produced by adding (in three steps, 10 min apart) a second diluent (250 mM Tris, 28 mM glucose, 104 mM citric acid, 12% egg yolk, 8% glycerol, 0.05% streptomycin, 500 UI penicillin/mL, and distilled water to 100 mL) at room temperature (20 8C) in a similar volume to the first dilution to result in a final concentration of 400 106 spermatozoa/mL, 12% egg yolk and 4% glycerol. Finally, the diluted semen was packaged in 0.5 mL plastic straws and the straws were sealed by plastic balls. The straws were placed into a cooler, being chilled from room temperature to 5 8C over 2 h; the straws were then kept for 2 h more at 5 8C, before starting the freezing process. After equilibration and just before the freezing protocols, aliquots of each sperm were taken for the assessment of sperm quality.
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2.1.4. Freezing and thawing of semen Fifteen semen freezes were performed in this experiment. Two different freezing protocols were tested: (1) straws that were frozen over the liquid nitrogen vapor for 15 min, 4 cm above the liquid nitrogen level, and were then plunged into and stored in liquid nitrogen (LN protocol), with the semen straws remaining in liquid nitrogen until the thawing period (2) straws that were moved directly from the cooler in a polystyrene box at 4 8C to the ultra-low temperature freezer at 152 8C (ULF protocol), where the semen straws remained stored in the ultrafreezer at 152 8C until thawing. Before freezing, the cooling velocity of the ultra-low temperature was defined. A probe (638 Pt, Crison Instruments, Barcelona) which was placed inside the straws filled with semen and diluents, showing a freezing rate of 4.5 8C/min from 5 to 10 8C and a faster freezing rate of 25 8C/min from 10 to 110 8C; from this temperature, the velocity of freezing was 10 8C/min from 110 to 140 8C. Thawing was carried out at 1, 30, 90 and 365 days after cryopreservation. The frozen straws were plunged into a water bath at 37 8C for 30 s, and then the semen was poured into 1 mL of PBS at 37 8C; the samples were then maintained at 37 8C until assessment. After thawing, the sperm motility and the percentages of acrosome intact spermatozoa and abnormal spermatozoa were determined as described previously. Fifteen straws per male were assessed each day (1, 30, 90 and 365) and in each freezing protocols (LN, ULF). 2.2. Experiment 2 2.2.1. Animals One hundred and fifty dairy goats of the Majorera breed (59 nulliparous and 91 multiparous) were used in this experiment. The goats showed regular 20–21 days estrous cycle before the start of the experiment, and an ultrasonography exam of the reproductive tract before the synchronization treatment confirmed that the females were not pseudo-pregnant. Feeding and housing of the goats were similar to that described previously for the bucks. 2.2.2. Estrus synchronization and heat detection Estrous cycles were synchronized by the insertion of intravaginal progestagen-impregnated sponges (45 mg fluorogestone acetate, Chrono-gest1, Intervet) for 11 days. Two days before the sponge withdrawal, the goats received an intramuscular injection of eCG (200 UI,
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Foligon1, Intervet) and 7.5 mg of a Prostaglandin-F2a analogue (Luprostiol, Prosolvin1, Intervet). Estrus detection was performed three times daily (8 h apart), starting 12 h after the removal of the sponge. Each goat was presented individually to the buck; the bucks were equipped with an apron to avoid penetration. 2.2.3. Experimental design and artificial insemination Ejaculates were collected from the three bucks that showed better freezability of semen in experiment 1. After collection, semen was pooled and processed as described previously to reach a final concentration of 400 106 spermatozoa/mL, 12% egg yolk and 4% glycerol. Thereafter, the diluted semen was packaged in straws (0.5 mL) and after cooling, the straws were frozen by the two freezing techniques (LN and ULF protocols). Before insemination, the semen straws were thawed in a water bath at 37 8C for 30 s. For each experimental group, the straws were assessed to confirm that the goats were inseminated with a minimum of 100 106 motile spermatozoa. The goats were assigned to four experimental groups according to age, date of previous kidding and daily milk production: in group LN-1 (n = 40), the goats were inseminated with thawed semen, previously frozen and stored in liquid nitrogen for 1 month; in group LN-6 (n = 35), the goats were inseminated with thawed semen, previously frozen and stored in liquid nitrogen for 6 months; in group ULF-1 (n = 40), the goats were inseminated with thawed semen, previously frozen and stored in an ultrafreezer at 152 8C for 1 month; and in group ULF-6 (n = 35), the goats were inseminated with thawed semen, previously frozen and stored in an ultrafreezer at 152 8C for 6 months. Artificial insemination was carried out between 18 and 20 h after the start of the estrous. The goats were inseminated via cervix with an insemination pipette (IMV technologies, L’ Aigle, France), using a speculum with a light source as described by Andersen et al. [23].
The semen was deposited as deeply as possible in the genital tract; all inseminations were performed by two experienced technicians. 2.2.4. Pregnancy diagnosis and fertility rate Pregnancy was confirmed by transabdominal ultrasound scanning on day 35 after mating using a real-time B portable scanner (Aloka SSD-500) equipped with a 5.0–7.5 MHz linear array transducer. The goats were considered pregnant when there was detection of the embryonic heartbeat [24]. Thereafter, the goats were reevaluated to check the fetal viability at 60, 90 and 120 days of pregnancy. Finally, after parturition, the kidding rate and prolificacy were recorded. 2.3. Statistical analysis Results are presented as mean standard error of the mean (SEM). Data of sperm viability from experiment 1 were analyzed using the general linear model procedure (ANOVA) of SPSS 10.0 (SPSS Inc., Chicago, IL, USA). The lineal model included the effects of the bucks (5 males), the freezing protocols (two protocols) and the time (1, 30, 90 and 365 days), as well as the interactions between them. Dependent variables expressed as percentages (sperm motility, acrosome integrity and abnormal sperm cells) were arcsine-transformed before analysis. Differences between means were analyzed by the Duncan test. The differences in the fertility results between the experimental groups, reproductive status of the goats (nulliparous/multiparous) and the effect of the time of cryopreservation were compared by Fisher’s exact test. Values were considered to be statistically significant when p < 0.05. 3. Results Table 1 presents the semen quality in the ejaculates of buck donors. No significant differences ( p > 0.1)
Table 1 Mean SEM of volume, sperm concentration and percentages of progressive sperm motility, acrosome integrity and abnormal sperm morphology in fresh semen of donor bucks. Buck
Volume (mL)
Sperm concentration (106 cells/mL)
Progressive sperm motility (%)
Abnormal spermatozoa (%)
Acrosome integrity (%)
1 2 3 4 5
1.7a 0.2 1.2b 0.1 1.1b 0.1 1.2b 0.2 1.1b 0.1
4.2a 0.5 6.1b 0.6 5.6b 0.3 4.9ab 0.5 5.2b 0.1
75.3 1.8 73.5 2.8 72.7 3.5 71.3 4.9 76.3 2.1
6.5 1.2 3.6 0.3 3.3 0.5 4.0 0.8 3.1 0.4
86.1 3.9 87.9 3.1 90.7 2.9 90.8 2.2 87.2 4.6
Different superscripts letters (a and b) within the same column denote significant differences ( p < 0.05).
M. Batista et al. / Theriogenology 71 (2009) 1307–1315
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Table 2 Percentages (mean SEM) of frozen-thawed semen progressive motility throughout the experimental period in liquid nitrogen (LN) and an ultrafreezer (ULF). Experimental periods
Freezing protocols
Buck 1
Buck 3
Buck 4
Buck 5
Day 1
LN ULF
38.2a 3.0 41.1a 2.2
Buck 2 42.6a 1.6 38.4a 2.1
29.6b 3.4 28.4b 3.2
41.8a 2.8 40.7a 5.0
40.2a 2.0 41.7a 2.1
Day 30
LN ULF
40.3a 3.2 42.2a 4.3
39.8a 2.7 41.3a 1.9
29.7b 3.5 28.9b 3.5
38.5a 4.0 43.7a 4.6
42.0a 2.2 43.5a 3.3
Day 90
LN ULF
41.5a 3.2 37.9a 1.3
45.1a 3.1 36.8ab 4.1
30.0b 1.5 29.3b 2.3
40.8a 2.4 42.8a 3.0
43.8a 2.0 44.2a 2.5
Day 365
LN ULF
37.2a 2.1 38.2a 3.1
44.7a 1.9 45.3a 2.2
28.8b 1.2 29.4b 1.3
39.5a 2.4 38.8a 2.4
* *
Different superscript letters (a and b) within the same row denote significant differences ( p < 0.05). * Lost data.
were found in the sperm motility (individual mean range: 84.7–89.9%) among bucks; all males showed very close mean values for the proportion of progressive sperm motility. In addition, the percentage of abnormal cells was lower than 9% in all males (range: 1.0–8.0%) and the acrosome membrane integrity always reached values higher than 82% in all ejaculates. The progressive sperm motility in each experimental group is shown in Table 2. Four bucks (males 1, 2, 4, 5) did not show any significant differences in the mean percentages of total sperm motility between both freezing protocols (range: 49.4–68.7% and 51.9– 65.0% for the LN and ULF protocols, respectively); in addition, the percentage of progressive sperm motility for any individual buck did not change significantly over the experimental period. However, male 3 showed a significant ( p < 0.01) reduction in the sperm motility (total and progressive) from day 1 onwards in both freezing protocols. When the individual bucks were compared, no significant
differences were detected between both freezing protocols throughout the tested period. Tables 3 and 4 show the percentages of acrosome membrane integrity and abnormal sperm cells, respectively. With regard to the acrosome status, within each male, no significant differences were detected between the freezing protocols (LN and ULF), showing very similar mean values on the tested days. In addition, when the bucks were compared between them, only male 3 presented a lower percentage ( p < 0.05) of acrosome membrane-intact spermatozoa than the others (day 30 post-freezing). The percentage of abnormal cells was relatively low in all males (range: 2.0–18.0), with no significant differences observed between either the freezing techniques or the bucks; in addition, 85.5% of the straws showed a percentage of abnormal spermatozoa under 12%. The interval from the sponge withdrawal until the onset of estrous was 29.3 2.1 h and 72.7% (109/150) of the goats presented estrus signals between 24 and
Table 3 Percentages (mean SEM) of frozen-thawed semen acrosome membrane integrity throughout the experimental period in liquid nitrogen (LN) and an ultrafreezer (ULF). Experimental periods
Freezing protocols
Buck 1
Buck 2
Buck 3
Buck 4
Buck 5
Day 1
LN ULF
69.2 2.4 72.0 4.2
67.7 3.4 70.8 3.3
68.9 1.1 67.3 4.1
73.8 2.4 70.3 3.1
71.6 3.6 69.8 4.1
Day 30
LN ULF
68.3ab 4.4 67.6a 3.7
69.2ab 4.3 72.2a 3.5
63.3b 1.2 56.1b 4.5
72.0a 2.5 72.9a 5.7
71.7a 2.2 68.3a 4.9
Day 90
LN ULF
70.1 5.5 70.6 3.1
69.0 2.5 69.3 2.5
66.9 1.1 67.1 2.0
68.3 9.9 71.8 6.6
73.4 4.9 67.8 9.2
Day 365
LN ULF
67.0 4.2 70.8 6.0
69.7 3.2 70.8 3.1
65.9 2.0 68.2 3.0
71.1 4.2 70.3 4.5
Different superscript letters (a and b) within the same row denote significant differences ( p < 0.05). * Lost data.
* *
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Table 4 Percentages (mean SEM) of frozen-thawed semen abnormal sperm cells throughout the experimental period in liquid nitrogen (LN) and an ultrafreezer (ULF). Experimental periods
*
Freezing protocols
Buck 1
Buck 2
Buck 3
Buck 4
Buck 5
Day 1
LN ULF
7.9 1.5 8.2 2.6
6.9 2.0 7.5 3.9
6.4 0.9 7.2 1.3
8.0 0.8 7.7 0.5
7.5 1.5 7.6 0.9
Day 30
LN ULF
7.7 1.6 8.6 2.5
6.7 0.5 9.2 1.7
6.6 1.1 7.4 1.2
8.1 0.7 7.5 1.0
6.9 2.2 8.5 2.0
Day 90
LN ULF
7.6 1.7 7.1 3.1
7.2 3.3 8.2 3.3
7.1 1.8 7.9 2.2
8.3 1.0 7.2 1.4
6.8 3.4 7.9 4.2
Day 365
LN ULF
7.3 1.7 8.3 1.8
8.0 2.6 7.4 0.9
7.1 3.2 7.6 2.7
8.5 1.5 7.0 1.2
* *
Lost data.
Table 5 Pregnancy rate and kidding rate after artificial insemination with frozen-thawed semen stored for 1 and 6 months in liquid nitrogen (LN) and an ultrafreezer (ULF). Freezing protocols a
LN-1 ULF-1b LN-6c ULF-6d a b c d
LN-1: liquid nitrogen ULF-1: ultrafreezer 1 LN-6: liquid nitrogen ULF-6: ultrafreezer 6
Pregnancy rate day 35 (%)
Pregnancy rate day 60 (%)
Kidding rate (%)
Prolificacy
45.0 40.0 42.8 40.0
45.0 40.0 42.8 40.0
43.6 38.5 42.8 40.0
1.64 1.67 1.60 1.64
(18/40) (16/40) (15/35) (14/35)
(18/40) (16/40) (15/35) (14/35)
(17/39) (15/39) (15/35) (14/35)
(28/17) (25/15) (24/15) (23/14)
1 month after preservation. month after preservation. 6 months after preservation. months after preservation.
32 h after sponge removal. In addition, the estrus length did not differ between experimental groups (36.8 1.1, 38.1 2.2, 37.2 2.7, 38.6 1.9, for groups LN-1, ULF-1, LN-6 and ULF-6, respectively). However, the estrus length was about 9 h longer in multiparous (41.2 h) than in nulliparous goats (32.0 h). Finally, Table 5 expresses the results of fertility after artificial insemination. The pregnancy rate was around 40% in both freezing protocols (LN and ULF), without
significant difference in the percentages of kidding when semen was used for insemination following 1 and 6 months of storage. In addition, no differences ( p > 0.05) were observed in prolificacy, with a similar percentage of single (31.2%, 10/32 vs 34.5%, 10/29 for LN vs ULF, respectively) and double bearing goats (68.7%, 20/32 vs 65.5%, 19/29 for LN vs ULF, respectively). However, as demonstrated in Table 6, the multiparous goats showed a significantly higher
Table 6 Kidding rate and prolificacy after artificial insemination with frozen-thawed semen in nulliparous and multiparous goats. Freezing protocols
a
LN-1 ULF-1b LN-6c ULF-6d
Kidding rate (%)
Prolificacy
Nulliparous
Multiparous
Nulliparous
Multiparous
33.3a 29.4a 33.3a 28.6a
50.0b 45.4b 47.8b 47.6b
1.20a 1.40a 1.25a 1.25a
1.83b 1.80b 1.73b 1.80b
(5/15) (5/17) (4/12) (4/14)
(12/24) (10/22) (11/23) (10/21)
(6/5) (7/5) (5/4) (5/4)
Different letters (a and b) within the same row and category (kidding rate/prolificacy) denote significant differences ( p < 0.05). a LN-1: liquid nitrogen 1 month after preservation. b ULF-1: ultrafreezer 1 month after preservation. c LN-6: liquid nitrogen 6 months after preservation. d ULF-6: ultrafreezer 6 months after preservation.
(21/12) (18/10) (19/11) (18/10)
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( p < 0.01) percentage of kidding than nulliparous females in both experimental periods (1 and 6 months of cryopreservation). Finally, the prolificacy was also higher ( p < 0.05) in multiparous than in nulliparous goats. 4. Discussion This study defines, for the first time, the in vitro quality of caprine semen after a long time (1 year) of cryopreservation using a 152 8C ultrafreezer. In addition, the results showed that long-term storage of the semen did not alter the semen quality. Finally, fertility trials confirmed that semen straws, frozen and stored in ultrafreezers at 152 8C, remained fertile after 6 months of cryopreservation. Fresh semen quality was very similar between males, and the only observed differences were in ejaculate volume and sperm concentration. After thawing, the sperm motility (total and progressive) showed similar mean values between both freezing protocols, comparable to that obtained in frozen-thawed semen of other caprine breeds [2,11,22,25–27]. Most previous studies have included the use of liquid nitrogen to freeze the semen straws [2,11,22] and a few studies have frozen the semen samples without liquid nitrogen [1,14]; however, all of these studies required liquid nitrogen for subsequent storage. A preliminary work [20] developed with a low number of animals proposed the use of ultralow freezers, where liquid nitrogen was not necessary for semen cryopreservation. In that study [20], the thawed semen showed mean values of progressive motility ranging between 31% and 42% (assessed subjectively) as compared to the values of 29–45% (assessed by CASA system), obtained in the present study. It seems evident that the ULF did not modify the parameters of sperm motility after freezing, showing highly correlated mean values between both protocols. The sperm motility was not modified throughout the experimental period in any individual buck, showing reasonably constant mean values for 1 year after freezing. These findings are in agreement with other published data reporting no variation of the seminal quality throughout the period of cryopreservation [28]; however, other studies have found deterioration in the viability of spermatozoa after long-term frozen storage [29,30]. On the other hand, the results of our study detected differences between bucks in the ability of the semen to remain viable after freezing. This individual variability in the sperm motility after freezing is relatively independent of fresh semen quality and it seems necessary to assess the seminal quality after
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freezing-thawing to define the donors bucks as ‘‘good freezers’’ or ‘‘bad freezers’’ [1]. The percentage of acrosome membrane integrity in both freezing protocols showed values (mean) around 60–70%. In other caprine breeds, after freezing and storing with LN, the acrosome membrane-intact spermatozoa ranged between 40% and 70% [11,12,22]. In addition, in semen samples assessed after cryopreservation with ULF, the acrosome integrity reached a mean value around 55% [20]. With regard to the percentage of abnormal sperm cells post-thawing, both freezing protocols (LN and ULF) showed no significant differences; in addition, in any individual male, this spermatic feature was similar over the test period and no significant differences were found when bucks were compared themselves. Different studies have assessed the percentage of normal sperm cells post-thawing and found a range between 50% and 80% after freezing with LN [2,9,11,12]; no results for abnormal sperm cells after freezing with the ULF protocol have been reported until present. According to our results, the ULF did not generate a higher percentage of sperm abnormalities than other freezing protocols. The kidding rates observed in our study (43.2% and 39.2% for LN and ULF, respectively) were similar to the fertility results in other caprine breeds after cervical insemination with frozen-thawed semen: 39% in the Cashmere goat [7], and 39–42% in Saanen goats [14]. Other studies reached higher fertility rates after artificial insemination with frozen-thawed semen: 47.6% in the Florida breed [12], 51% in Angora goat [30], 57% in the Murciano-Granadina goats [9] and 57–61% in Saanen and Alpine goats [1]. However, most of these studies were carried out with multiparous goats and our results showed a remarkable influence of the number of previous parities on the fertility rates. Nulliparous goats showed a significantly lower kidding rate (28–33%) than the multiparous goats (45–50%). Dorado et al. [12] found a higher fertility rate in multiparous (58.8%) than in uniparous goats (0%) after artificial insemination with semen frozen-thawed; in addition, it has been shown that after hormonal induction of estrus, nulliparous goat were less fertile than multiparous goats when inseminated intracervically [1]. The lower fertility rates observed in nulliparous goats could be explained by the time of insemination relative to ovulation; in multiparous goats, the insemination took place around the middle of the estrous period (estrus length: 41.2 h), while in nulliparous goats, the inseminations were carried out in the second stage of estrus (estrus length: 32.0 h). A higher fertility rate has been demonstrated in goats inseminated in the first stage of the estrus period [1]. On the other
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hand, it should be noted that the nulliparous goats showed higher degree of nervousness and the stress involved in the insemination technique could have generated the lower fertility rate observed in our study. Different studies have assessed different factors affecting fertility rates after artificial insemination with frozen-thawed semen [1,9,12]. It has been found that the fertility was better when the semen was collected in the breeding season, rather than in the non-breeding season [31]; in our study, semen collection was carried out during the breeding season. Consequently, the seasonal influence on sperm freezability and, thus, on fertility should be ruled out. In addition, the semen was pooled before freezing to avoid the influence for differences between bucks in the ability of the semen to remain viable following freezing [1]; moreover, the semen straws were assessed before insemination and only semen straws with a minimum requirement of progressive motility were used for AI. On the other hand, the influence of the technician could be ruled out since both inseminators had the same experience and no differences in fertility were detected between technicians. Finally, different studies have established the relationship between the site of semen deposition and the fertility rates [5,6,31], showing a higher percentage of fertility rate when the semen is deposited into the cervix or into the uterus than if the AI was vaginal. In our study, most of the inseminations (90%) were performed into the cervix. Few studies that have reported fertility rates after AI with frozen-thawed semen have valued the influence of the storage time of the semen straws on the fertility. Fougner [32] obtained a 63.4% kidding rate after intracervical inseminations with semen stored in LN for 1–3 years and no significant differences were found in fertility rates throughout the experimental period. However, other study has detected a decline in the seminal quality (in vitro) after storing the semen for 6 months in LN [6]. In our study, no differences were observed in the kidding rate after artificial insemination with semen frozen-thawed after 1 or 6 months of storage. Therefore, these results confirmed that the storage time of frozen semen had no influence on the subsequent fertility. Before this study, only one work [20] had proposed the use of the ULF to freeze and store caprine semen, but that study was carried out for a short storage time (2 months) and no fertility trials were performed. In our study, no differences were observed between both freezing techniques, presenting similar results of sperm quality in vitro throughout the experimental period (1 year). In addition, the fertility results confirmed that caprine semen remained fertile after cryopreservation
with the ULF, showing a kidding rate and prolificacy practically equal to that observed after AI with semen frozen with LN. Ultra-low temperature freezers have advantages over freezing with liquid nitrogen. It has been reported [18,19] that the higher storage capacity of the ULF, the lower time elapsed to freeze the straws after equilibration and the sample handling is easier than with LN; in addition, unlike cryopreservation with liquid nitrogen, periodic reposition is not required, and the global cost of the freezing protocol is cheaper. However, an important disadvantage of the ULF protocol is the limited mobility of the freezer that could reduce the efficacy of the AI in farms far from the lab. To avoid this limitation, at present, new research lines are developing, where semen straws frozen and stored in the ULF will be moved to liquid nitrogen to subsequently test the semen quality (in vitro and in vivo). In addition, further studies are needed to assess the fertility of caprine semen frozen using ULF after storing the semen for several years. References [1] Leboeuf B, Restall B, Salamon S. Production and storage of goat semen for artificial insemination. Anim Reprod Sci 2000;62:113–41. [2] Hidalgo M, Rodrı´guez I, Dorado JM. The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability. Anim Reprod Sci 2007;100:61–72. [3] Roca J, Carrizosa JA, Campos I, Lafuente A, Va´zquez JM, Martı´nez E. Viability and fertility of unwashed Murciano-Granadina goat spermatozoa diluted in Tris-egg yolk extender and stored at 5 8C. Small Ruminant Res 1997;25:147–53. [4] Leboeuf B, Forgerit Y, Bernelas D, Pougnard JL, Senty E, Driancourt MA. Efficacy of two types of vaginal sponges to control onset of oestrus, time of preovulatory LH peak and kidding rate in goats inseminated with variable numbers of spermatozoa. Theriogenology 2003;60:1371–8. [5] Paulenz H, So¨derquist L, Adnoy T, Soltun K, Saether PA, Fjellsoy KR, et al. Effect of cervical and vaginal insemination with liquid semen stored at room temperature on fertility of goats. Anim Reprod Sci 2005;86:109–17. [6] Ritar AJ, Salamon S. Fertility of fresh and frozen-thawed semen of the Angora goat. Aust J Biol Sci 1983;36:49–59. [7] Ritar AJ, Ball PD, O’May PJ. Artificial insemination of Cashmere goats: effects on fertility and fecundity of intravaginal treatment, method and time of insemination, semen freezing process, number of motile spermatozoa and age of females. Reprod Fertil Dev 1990;2:377–84. [8] Karatzas G, Karagiannidis A, Varsakeli S, Brikas P. Fertility of fresh and frozen-thawed goat semen during the nonbreeding season. Theriogenology 1997;48:1049–59. [9] Salvador I, Viudes-de-Castro MP, Bernacer J, Go´mez EA, Silvestre MA. Factors affecting pregnancy rate in artificial insemination with frozen semen during non-breeding season in Murciano-Granadina goats: a field assay. Reprod Domest Anim 2005;40:526–9.
M. Batista et al. / Theriogenology 71 (2009) 1307–1315 [10] Baril G, Leboeuf B, Saumande J. Synchronization of estrus in goats: the relationship between time of occurrence of estrus and fertility following artificial insemination. Theriogenology 1993;40:621–8. [11] Singh MP, Sinha AK, Singh BK. Effect of cryoprotectants on certain seminal attributes and on the fertility of buck spermatozoa. Theriogenology 1995;43:1047–53. [12] Dorado J, Rodrı´guez I, Hidalgo M. Cryopreservation of goat spermatozoa: comparison of two freezing extenders base on post-thaw sperm quality and fertility rates after artificial insemination. Theriogenology 2007;68:168–77. [13] Dauzier L. Artificial insemination in the goat. In: Dalling, editor. Encyclopedia of Veterinary Medicine. W. Green and Soon; 1966. p. 269–71. [14] Gacitua H, Arav A. Successful pregnancies with directional freezing of large volume buck semen. Theriogenology 2005;63:931–8. [15] Kaabi M, Alvarez M, Anel E, Chamorro C, Boixo JC, Paz P, et al. Influence of breed and age on morphometry and depth of inseminating catheter penetration in the ewe cervix: a postmortem study. Theriogenology 2006;66:1876–83. [16] Eppleston J, Salamon S, Moore N, Evans G. The depth of cervical insemination and site of intrauterine insemination and their relationship to the fertility of frozen-thawed ram semen. Anim Reprod Sci 1994;36:211–25. [17] Holt W. Alternative strategies for the long-term preservation of spermatozoa. Reprod Fertil Dev 1997;9:309–19. [18] Alamo D, Batista M, Gonza´lez F, Rodrı´guez N, Cruz G, Cabrera F, et al. Cryopreservation of semen in the dog: use of ultrafreezers of 152 8C as a viable alternative to liquid nitrogen. Theriogenology 2005;63:72–82. [19] Batista M, Alamo D, Gonza´lez F, Cruz MG, Gracia A. Influence of the freezing technique (nitrogen liquid vs ultrafreezer of 152 8C) and male-to-male variation over the semen quality in Canarian Mastiff breed dogs. Reprod Domest Anim 2006;41:423–8. [20] Medrano A, Cabrera F, Gonza´lez F, Batista M, Gracia A. Is sperm cryopreservation at 150 degree C a feasible alternative? Cryoletters 2002;23:167–72.
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[21] Barth AD, Oko RJ. Abnormal Morphology of Bovine Spermatozoa. Ames, IA: Iowa State University Press; 1989. [22] Cabrera F, Gonza´lez F, Batista M, Calero P, Medrano A, Gracia A. The effect of removal of seminal plasma, egg yolk level and season on sperm freezability of Canary buck (Capra hircus). Reprod Domest Anim 2005;40:191–5. [23] Andersen K, Aamdal J, Fougner JA. Intrauterine and deep cervical insemination with frozen semen in sheep. Zuchthygiene 1973;8:113–8. [24] Martinez MF, Bosch P, Bosch RA. Determination of early pregnancy and embryonic growth in goats by transrectal ultrasound scanning. Theriogenology 1998;49:1555–65. [25] Tuli RK, Holtz W. Effect of season on the freezability of Boer goat semen in the northern temperate zone. Theriogenology 1995;43:1359–63. [26] Tuli RK, Holtz W. Effect of glycerolization procedure and removal of seminal plasma on post-thaw survival and gotrelease from Boer goat spermatozoa. Theriogenology 1994;42:547–55. [27] Chang-Yong C, Jung-Gon K, Sang-Rae C, Dong-Soo S, YoungKeun K, Balasubramanian S, et al. Influence of seasons, extenders, slow and rapid freezing on seminal characters in Korean native bucks. Reprod Domest Anim 2006;41:55–60. [28] Waide Y, Niwa T, Asanuma R. Studies on the preservation of liquid and frozen semen of domestic animals: III. Viability and fertility of frozen goat spermatozoa. Jpn J Anim Reprod 1977;23:129–37. [29] Corteel JM. Effect du ‘lavage’ sur la conservation des spermatozoides de bouc a base temperatura. Ann Biol Anim Biochim Biophys 1975;15:525–8. [30] Ritar AJ, Salamon S. Effects of month of collection, method of processing, concentration of egg yolk and duration of frozen storage on viability of Angora goat spermatozoa. Small Ruminant Res 1991;4:29–37. [31] Corteel JM, Leboeuf B, Baril G. Artificial breeding of adult goats and kids induced with hormones to ovulate outside the breeding season. Small Ruminant Res 1988;1:19–33. [32] Fougner JA. Die intrauterine besamung der ziege mit tiefgefrorenem sperma. Zuchthygiene 1979;14:104–10.
Theriogenology 71 (2009) 1307–1315 www.theriojournal.com
Successful artificial insemination using semen frozen and stored by an ultrafreezer in the Majorera goat breed M. Batista a,*, T. Nin˜o a, D. Alamo a, N. Castro b, M. Santana a, F. Gonza´lez a, F. Cabrera a, A. Gracia a a
Obstetrics and Reproduction, Faculty of Veterinary of Las Palmas, Transmontan˜a s/n, 35413 Arucas, Spain b Animal Science, Faculty of Veterinary of Las Palmas, Transmontan˜a s/n, 35413 Arucas, Spain Received 27 October 2008; received in revised form 18 December 2008; accepted 29 December 2008
Abstract The semen of five Majorera breed bucks was collected and processed to reach a final concentration of 200 106 spermatozoa/ straw in the extender containing 4% of glycerol and 12% of egg yolk. Two freezing techniques were assessed: (LN) straws were frozen and stored in liquid nitrogen, and (ULF) straws were frozen and stored in the ultra-low freezer at 152 8C. Semen quality (sperm motility, acrosome integrity and abnormal sperm cells percentages) was determined for different storage times (1, 30, 90 and 365 days of cryopreservation). Thereafter, 150 Majorera goats were assigned to four experimental groups: for groups LN-1 (n = 40) and LN-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in liquid nitrogen, respectively, while for groups ULF-1 (n = 40) and ULF-6 (n = 35), the goats were transcervically inseminated with frozenthawed semen stored for 1 and 6 months in an ultra-low freezer at 152 8C, respectively. The pregnancy rate was determined by transabdominal ultrasound scanning; in addition, the kidding rate and prolificacy were recorded at parturition. In vitro results showed that the freezing protocol did not affect sperm quality with similar values for up to 1 year of cryopreservation. The kidding rates were not significantly different between experimental groups (43.6%, 38.5%, 42.8% and 40.0% for groups LN-1, ULF-1, LN-6 and ULF-6, respectively). In all experimental groups, the kidding rate and prolificacy were significantly higher ( p < 0.01) in multiparous than in nulliparous goats. Therefore, the in vitro results and fertility trials confirmed the efficiency of the ULF technique for freezing and storage of goat semen. # 2009 Elsevier Inc. All rights reserved. Keywords: Goat; Semen; Ultrafreezer; Fertility; Long-term storage
1. Introduction Artificial insemination (AI) with fresh, chilled or frozen-thawed semen is a basic tool in goat breeding, allowing the diffusion of caprine semen with high * Corresponding author at: Unidad de Reproduccio´n y Obstetricia, Facultad de Veterinaria de Las Palmas, Transmontan˜a s/n, 35413 Arucas, Las Palmas, Spain. Tel.: +34 92 8454356; fax: +34 92 8454301. E-mail address: [email protected] (M. Batista). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.12.024
genetic value [1,2]. In recent years, artificial insemination with fresh semen has become a common technique in goats [3–5]; however, the commercial use of frozenthawed semen has been relatively limited in caprine reproduction. As in other domestic animals, the freezing process reduces the viability of the caprine spermatozoa; kidding rate after AI with frozen-thawed semen ranged between 30% and 70% [4,6–12], while with fresh or chilled semen produces values between 60% and 80% [3,5,6,13]. With transcervical insemination, a
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low proportion of spermatozoa traverse the cervical canal, meaning a relatively higher number of sperm cells are necessary to get reasonable pregnancy rates [3,10,14]. Different studies have noted this fact as a handicap in artificial insemination in small ruminants [14,15]. Laparoscopic intrauterine insemination of goats resulted in improved fertility rates [7,16]; however, the high costs associated with this technique and the necessary qualified inseminators are restrictive factors for the use of laparoscopy to inseminate goats. The cryopreservation of semen straws in liquid nitrogen (LN) is the standard protocol to freeze and store caprine semen. Other studies have described the freezing of semen by placing pellets in dry ice at 79 8C, but these require the subsequent immersion of the pellets in LN for the frozen storage of semen [1]. In some areas, LN is not always readily available and its cost is relatively high. The development of freezedrying and vitrification methods, avoiding the use of LN, has been postulated [17] as a feasible alternative for sperm cryopreservation. However, in vivo trials have not been developed to confirm the effectiveness of these alternative strategies. A recent study [14] in goats did report high fertility rates after artificial insemination with semen frozen using a directional freezing machine; however, this study also used LN for the storage of semen. In canine, different studies [18,19] have confirmed that the use of ultrafreezers at 152 8C for freezing and storing canine semen could represent a potential alternative to liquid nitrogen. Our preliminary study [20] developed in Majorera bucks assessed, for the first time, the use of ultrafreezers at 152 8C for freezing and storing caprine semen. This study showed that the in vitro seminal quality (2 months after freezing) was not significantly different for seminal straws frozen and stored in liquid nitrogen when compared with those frozen and stored exclusively in the ultra-low temperature freezer at 152 8C. However, the results of longterm storage of semen frozen using ultrafreezers at 152 8C have not been related; in addition, fertility trials to test the fertilizing capacity of semen frozen by an ultrafreezer at 152 8C has not been developed until now. The aim of this experiment was to assess the use of an ultra-low freezer at 152 8C both for freezing and for short-term (1, 3 months) and long-term (12 months) storage of caprine semen. In addition, in vivo trials were carried out to confirm the caprine semen remained fertile after cryopreservation with the ultra-low temperature freezer at 152 8C.
2. Materials and methods 2.1. Experiment 1 2.1.1. Animals Five healthy bucks of the Majorera breed, aged between 3.5 and 5 years, were selected by their breeding value. The study was conducted at the experimental farm of the Veterinary Faculty of Las Palmas (Canary Islands, Spain, 288N, 238W). The animals were housed in a collective pen, and fed was a mix of maize and bran (400 g per male) and dehydrated alfalfa; mineral salts and water were provided ‘‘ad libitum’’. All experimental work was carried out according the Spanish laws for animal research and experimentation. 2.1.2. Semen collection and evaluation During the breeding season (September–November 2006), semen was collected from each male, at 2 per week for 5 weeks, using an artificial vagina. Immediately after collection, the ejaculates were placed in a water bath (37 8C) and aliquots were taken for the assessment of semen quality. The sperm concentration, sperm motility, and the percentages of sperm cells with intact acrosome membrane integrity and with abnormal morphology were defined. The semen volume was recorded directly in a calibrated tube. The sperm concentration was determined with a Spermacue1 spectrophotometer (Minitu¨b GmbH, Germany), which had been calibrated for buck semen. Before analysis, 25 mL of each semen sample was added to 5 mL plastic tubes containing 975 mL of PBS at 37 8C, and, this solution was then, maintained at 37 8C until assessment. The proportion of motile sperm cells was evaluated subjectively using a phase contrast microscope (magnification: 1000) and with a Computer Assisted Sperm Analysis (CASA) system (Sperm Vision Lite1, Minitu¨b Ibe´rica, Tarragona, Spain); the semen variables included in the analysis were the percentage of total motile spermatozoa, percentage of progressive fast spermatozoa, the percentage of locally motile spermatozoa and the percentage of non-motile sperm cells. For each diluted semen sample, two aliquots of 10 mL were placed on a Makler chamber at 37 8C and the sperm contained in seven randomized fields in each of the two aliquots were assessed for motility; at least, 2000 sperm cells/aliquot were evaluated to define the motility parameters. The settings of the motility analyzer were as follows: number of frames, 16; frames per second, 25; velocity limit for non-motile sperm cells, 10 mm/s; velocity limit for locally motile
M. Batista et al. / Theriogenology 71 (2009) 1307–1315
sperm cells, 20 mm/s; velocity limit for low motile sperm cells, 30 mm/s; velocity limit for medium motile sperm cells, 60 mm/s; filter for particles, 20%. The acrosome integrity was assessed using Spermac staining technique (Minitu¨b, Tiefenbach, Germany) counting a minimum of 200 sperm cells per slide (phase contrast microscopy, at 1000). The percentage of cells with abnormal morphology was estimated by placing 50 mL of the semen sample on a pre-warmed (37 8C) slide; after staining with the Blom’s eosinnigrosin stain [21], the samples were assessed under a phase contrast microscopy (100) by examining 200 cells per sample. Only semen samples with progressive sperm motility >75%, acrosome intact spermatozoa >80% and normal sperm morphology >85% were used. 2.1.3. Semen processing Between 5 and 10 min after collection, the ejaculates from each male were washed to remove the seminal plasma. The ejaculate was diluted in washing solution (250 mM Tris, 28 mM glucose, 104 mM citric acid, 0.05% streptomycin and 500 UI penicillin/mL) at 37 8C, and centrifuged twice at 700 g for 15 min (room temperature, 20 8C) and the supernatant was removed. The ejaculates of each buck were processed individually. Semen processing was performed as described by Cabrera et al. [22]. Briefly, the sperm pellet was diluted (1:3) in a Tris-based freezing medium (250 mM Tris, 28 mM glucose, 104 mM citric acid, 12% egg yolk, 0.05% streptomycin, 500 UI penicillin/mL, and distilled water to 100 mL). Thereafter, the sperm concentration was determined and the volume was adjusted (at room temperature, 20 8C) to reach a final concentration of 800 106 spermatozoa/mL. After 5 min, a new dilution was produced by adding (in three steps, 10 min apart) a second diluent (250 mM Tris, 28 mM glucose, 104 mM citric acid, 12% egg yolk, 8% glycerol, 0.05% streptomycin, 500 UI penicillin/mL, and distilled water to 100 mL) at room temperature (20 8C) in a similar volume to the first dilution to result in a final concentration of 400 106 spermatozoa/mL, 12% egg yolk and 4% glycerol. Finally, the diluted semen was packaged in 0.5 mL plastic straws and the straws were sealed by plastic balls. The straws were placed into a cooler, being chilled from room temperature to 5 8C over 2 h; the straws were then kept for 2 h more at 5 8C, before starting the freezing process. After equilibration and just before the freezing protocols, aliquots of each sperm were taken for the assessment of sperm quality.
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2.1.4. Freezing and thawing of semen Fifteen semen freezes were performed in this experiment. Two different freezing protocols were tested: (1) straws that were frozen over the liquid nitrogen vapor for 15 min, 4 cm above the liquid nitrogen level, and were then plunged into and stored in liquid nitrogen (LN protocol), with the semen straws remaining in liquid nitrogen until the thawing period (2) straws that were moved directly from the cooler in a polystyrene box at 4 8C to the ultra-low temperature freezer at 152 8C (ULF protocol), where the semen straws remained stored in the ultrafreezer at 152 8C until thawing. Before freezing, the cooling velocity of the ultra-low temperature was defined. A probe (638 Pt, Crison Instruments, Barcelona) which was placed inside the straws filled with semen and diluents, showing a freezing rate of 4.5 8C/min from 5 to 10 8C and a faster freezing rate of 25 8C/min from 10 to 110 8C; from this temperature, the velocity of freezing was 10 8C/min from 110 to 140 8C. Thawing was carried out at 1, 30, 90 and 365 days after cryopreservation. The frozen straws were plunged into a water bath at 37 8C for 30 s, and then the semen was poured into 1 mL of PBS at 37 8C; the samples were then maintained at 37 8C until assessment. After thawing, the sperm motility and the percentages of acrosome intact spermatozoa and abnormal spermatozoa were determined as described previously. Fifteen straws per male were assessed each day (1, 30, 90 and 365) and in each freezing protocols (LN, ULF). 2.2. Experiment 2 2.2.1. Animals One hundred and fifty dairy goats of the Majorera breed (59 nulliparous and 91 multiparous) were used in this experiment. The goats showed regular 20–21 days estrous cycle before the start of the experiment, and an ultrasonography exam of the reproductive tract before the synchronization treatment confirmed that the females were not pseudo-pregnant. Feeding and housing of the goats were similar to that described previously for the bucks. 2.2.2. Estrus synchronization and heat detection Estrous cycles were synchronized by the insertion of intravaginal progestagen-impregnated sponges (45 mg fluorogestone acetate, Chrono-gest1, Intervet) for 11 days. Two days before the sponge withdrawal, the goats received an intramuscular injection of eCG (200 UI,
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Foligon1, Intervet) and 7.5 mg of a Prostaglandin-F2a analogue (Luprostiol, Prosolvin1, Intervet). Estrus detection was performed three times daily (8 h apart), starting 12 h after the removal of the sponge. Each goat was presented individually to the buck; the bucks were equipped with an apron to avoid penetration. 2.2.3. Experimental design and artificial insemination Ejaculates were collected from the three bucks that showed better freezability of semen in experiment 1. After collection, semen was pooled and processed as described previously to reach a final concentration of 400 106 spermatozoa/mL, 12% egg yolk and 4% glycerol. Thereafter, the diluted semen was packaged in straws (0.5 mL) and after cooling, the straws were frozen by the two freezing techniques (LN and ULF protocols). Before insemination, the semen straws were thawed in a water bath at 37 8C for 30 s. For each experimental group, the straws were assessed to confirm that the goats were inseminated with a minimum of 100 106 motile spermatozoa. The goats were assigned to four experimental groups according to age, date of previous kidding and daily milk production: in group LN-1 (n = 40), the goats were inseminated with thawed semen, previously frozen and stored in liquid nitrogen for 1 month; in group LN-6 (n = 35), the goats were inseminated with thawed semen, previously frozen and stored in liquid nitrogen for 6 months; in group ULF-1 (n = 40), the goats were inseminated with thawed semen, previously frozen and stored in an ultrafreezer at 152 8C for 1 month; and in group ULF-6 (n = 35), the goats were inseminated with thawed semen, previously frozen and stored in an ultrafreezer at 152 8C for 6 months. Artificial insemination was carried out between 18 and 20 h after the start of the estrous. The goats were inseminated via cervix with an insemination pipette (IMV technologies, L’ Aigle, France), using a speculum with a light source as described by Andersen et al. [23].
The semen was deposited as deeply as possible in the genital tract; all inseminations were performed by two experienced technicians. 2.2.4. Pregnancy diagnosis and fertility rate Pregnancy was confirmed by transabdominal ultrasound scanning on day 35 after mating using a real-time B portable scanner (Aloka SSD-500) equipped with a 5.0–7.5 MHz linear array transducer. The goats were considered pregnant when there was detection of the embryonic heartbeat [24]. Thereafter, the goats were reevaluated to check the fetal viability at 60, 90 and 120 days of pregnancy. Finally, after parturition, the kidding rate and prolificacy were recorded. 2.3. Statistical analysis Results are presented as mean standard error of the mean (SEM). Data of sperm viability from experiment 1 were analyzed using the general linear model procedure (ANOVA) of SPSS 10.0 (SPSS Inc., Chicago, IL, USA). The lineal model included the effects of the bucks (5 males), the freezing protocols (two protocols) and the time (1, 30, 90 and 365 days), as well as the interactions between them. Dependent variables expressed as percentages (sperm motility, acrosome integrity and abnormal sperm cells) were arcsine-transformed before analysis. Differences between means were analyzed by the Duncan test. The differences in the fertility results between the experimental groups, reproductive status of the goats (nulliparous/multiparous) and the effect of the time of cryopreservation were compared by Fisher’s exact test. Values were considered to be statistically significant when p < 0.05. 3. Results Table 1 presents the semen quality in the ejaculates of buck donors. No significant differences ( p > 0.1)
Table 1 Mean SEM of volume, sperm concentration and percentages of progressive sperm motility, acrosome integrity and abnormal sperm morphology in fresh semen of donor bucks. Buck
Volume (mL)
Sperm concentration (106 cells/mL)
Progressive sperm motility (%)
Abnormal spermatozoa (%)
Acrosome integrity (%)
1 2 3 4 5
1.7a 0.2 1.2b 0.1 1.1b 0.1 1.2b 0.2 1.1b 0.1
4.2a 0.5 6.1b 0.6 5.6b 0.3 4.9ab 0.5 5.2b 0.1
75.3 1.8 73.5 2.8 72.7 3.5 71.3 4.9 76.3 2.1
6.5 1.2 3.6 0.3 3.3 0.5 4.0 0.8 3.1 0.4
86.1 3.9 87.9 3.1 90.7 2.9 90.8 2.2 87.2 4.6
Different superscripts letters (a and b) within the same column denote significant differences ( p < 0.05).
M. Batista et al. / Theriogenology 71 (2009) 1307–1315
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Table 2 Percentages (mean SEM) of frozen-thawed semen progressive motility throughout the experimental period in liquid nitrogen (LN) and an ultrafreezer (ULF). Experimental periods
Freezing protocols
Buck 1
Buck 3
Buck 4
Buck 5
Day 1
LN ULF
38.2a 3.0 41.1a 2.2
Buck 2 42.6a 1.6 38.4a 2.1
29.6b 3.4 28.4b 3.2
41.8a 2.8 40.7a 5.0
40.2a 2.0 41.7a 2.1
Day 30
LN ULF
40.3a 3.2 42.2a 4.3
39.8a 2.7 41.3a 1.9
29.7b 3.5 28.9b 3.5
38.5a 4.0 43.7a 4.6
42.0a 2.2 43.5a 3.3
Day 90
LN ULF
41.5a 3.2 37.9a 1.3
45.1a 3.1 36.8ab 4.1
30.0b 1.5 29.3b 2.3
40.8a 2.4 42.8a 3.0
43.8a 2.0 44.2a 2.5
Day 365
LN ULF
37.2a 2.1 38.2a 3.1
44.7a 1.9 45.3a 2.2
28.8b 1.2 29.4b 1.3
39.5a 2.4 38.8a 2.4
* *
Different superscript letters (a and b) within the same row denote significant differences ( p < 0.05). * Lost data.
were found in the sperm motility (individual mean range: 84.7–89.9%) among bucks; all males showed very close mean values for the proportion of progressive sperm motility. In addition, the percentage of abnormal cells was lower than 9% in all males (range: 1.0–8.0%) and the acrosome membrane integrity always reached values higher than 82% in all ejaculates. The progressive sperm motility in each experimental group is shown in Table 2. Four bucks (males 1, 2, 4, 5) did not show any significant differences in the mean percentages of total sperm motility between both freezing protocols (range: 49.4–68.7% and 51.9– 65.0% for the LN and ULF protocols, respectively); in addition, the percentage of progressive sperm motility for any individual buck did not change significantly over the experimental period. However, male 3 showed a significant ( p < 0.01) reduction in the sperm motility (total and progressive) from day 1 onwards in both freezing protocols. When the individual bucks were compared, no significant
differences were detected between both freezing protocols throughout the tested period. Tables 3 and 4 show the percentages of acrosome membrane integrity and abnormal sperm cells, respectively. With regard to the acrosome status, within each male, no significant differences were detected between the freezing protocols (LN and ULF), showing very similar mean values on the tested days. In addition, when the bucks were compared between them, only male 3 presented a lower percentage ( p < 0.05) of acrosome membrane-intact spermatozoa than the others (day 30 post-freezing). The percentage of abnormal cells was relatively low in all males (range: 2.0–18.0), with no significant differences observed between either the freezing techniques or the bucks; in addition, 85.5% of the straws showed a percentage of abnormal spermatozoa under 12%. The interval from the sponge withdrawal until the onset of estrous was 29.3 2.1 h and 72.7% (109/150) of the goats presented estrus signals between 24 and
Table 3 Percentages (mean SEM) of frozen-thawed semen acrosome membrane integrity throughout the experimental period in liquid nitrogen (LN) and an ultrafreezer (ULF). Experimental periods
Freezing protocols
Buck 1
Buck 2
Buck 3
Buck 4
Buck 5
Day 1
LN ULF
69.2 2.4 72.0 4.2
67.7 3.4 70.8 3.3
68.9 1.1 67.3 4.1
73.8 2.4 70.3 3.1
71.6 3.6 69.8 4.1
Day 30
LN ULF
68.3ab 4.4 67.6a 3.7
69.2ab 4.3 72.2a 3.5
63.3b 1.2 56.1b 4.5
72.0a 2.5 72.9a 5.7
71.7a 2.2 68.3a 4.9
Day 90
LN ULF
70.1 5.5 70.6 3.1
69.0 2.5 69.3 2.5
66.9 1.1 67.1 2.0
68.3 9.9 71.8 6.6
73.4 4.9 67.8 9.2
Day 365
LN ULF
67.0 4.2 70.8 6.0
69.7 3.2 70.8 3.1
65.9 2.0 68.2 3.0
71.1 4.2 70.3 4.5
Different superscript letters (a and b) within the same row denote significant differences ( p < 0.05). * Lost data.
* *
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M. Batista et al. / Theriogenology 71 (2009) 1307–1315
Table 4 Percentages (mean SEM) of frozen-thawed semen abnormal sperm cells throughout the experimental period in liquid nitrogen (LN) and an ultrafreezer (ULF). Experimental periods
*
Freezing protocols
Buck 1
Buck 2
Buck 3
Buck 4
Buck 5
Day 1
LN ULF
7.9 1.5 8.2 2.6
6.9 2.0 7.5 3.9
6.4 0.9 7.2 1.3
8.0 0.8 7.7 0.5
7.5 1.5 7.6 0.9
Day 30
LN ULF
7.7 1.6 8.6 2.5
6.7 0.5 9.2 1.7
6.6 1.1 7.4 1.2
8.1 0.7 7.5 1.0
6.9 2.2 8.5 2.0
Day 90
LN ULF
7.6 1.7 7.1 3.1
7.2 3.3 8.2 3.3
7.1 1.8 7.9 2.2
8.3 1.0 7.2 1.4
6.8 3.4 7.9 4.2
Day 365
LN ULF
7.3 1.7 8.3 1.8
8.0 2.6 7.4 0.9
7.1 3.2 7.6 2.7
8.5 1.5 7.0 1.2
* *
Lost data.
Table 5 Pregnancy rate and kidding rate after artificial insemination with frozen-thawed semen stored for 1 and 6 months in liquid nitrogen (LN) and an ultrafreezer (ULF). Freezing protocols a
LN-1 ULF-1b LN-6c ULF-6d a b c d
LN-1: liquid nitrogen ULF-1: ultrafreezer 1 LN-6: liquid nitrogen ULF-6: ultrafreezer 6
Pregnancy rate day 35 (%)
Pregnancy rate day 60 (%)
Kidding rate (%)
Prolificacy
45.0 40.0 42.8 40.0
45.0 40.0 42.8 40.0
43.6 38.5 42.8 40.0
1.64 1.67 1.60 1.64
(18/40) (16/40) (15/35) (14/35)
(18/40) (16/40) (15/35) (14/35)
(17/39) (15/39) (15/35) (14/35)
(28/17) (25/15) (24/15) (23/14)
1 month after preservation. month after preservation. 6 months after preservation. months after preservation.
32 h after sponge removal. In addition, the estrus length did not differ between experimental groups (36.8 1.1, 38.1 2.2, 37.2 2.7, 38.6 1.9, for groups LN-1, ULF-1, LN-6 and ULF-6, respectively). However, the estrus length was about 9 h longer in multiparous (41.2 h) than in nulliparous goats (32.0 h). Finally, Table 5 expresses the results of fertility after artificial insemination. The pregnancy rate was around 40% in both freezing protocols (LN and ULF), without
significant difference in the percentages of kidding when semen was used for insemination following 1 and 6 months of storage. In addition, no differences ( p > 0.05) were observed in prolificacy, with a similar percentage of single (31.2%, 10/32 vs 34.5%, 10/29 for LN vs ULF, respectively) and double bearing goats (68.7%, 20/32 vs 65.5%, 19/29 for LN vs ULF, respectively). However, as demonstrated in Table 6, the multiparous goats showed a significantly higher
Table 6 Kidding rate and prolificacy after artificial insemination with frozen-thawed semen in nulliparous and multiparous goats. Freezing protocols
a
LN-1 ULF-1b LN-6c ULF-6d
Kidding rate (%)
Prolificacy
Nulliparous
Multiparous
Nulliparous
Multiparous
33.3a 29.4a 33.3a 28.6a
50.0b 45.4b 47.8b 47.6b
1.20a 1.40a 1.25a 1.25a
1.83b 1.80b 1.73b 1.80b
(5/15) (5/17) (4/12) (4/14)
(12/24) (10/22) (11/23) (10/21)
(6/5) (7/5) (5/4) (5/4)
Different letters (a and b) within the same row and category (kidding rate/prolificacy) denote significant differences ( p < 0.05). a LN-1: liquid nitrogen 1 month after preservation. b ULF-1: ultrafreezer 1 month after preservation. c LN-6: liquid nitrogen 6 months after preservation. d ULF-6: ultrafreezer 6 months after preservation.
(21/12) (18/10) (19/11) (18/10)
M. Batista et al. / Theriogenology 71 (2009) 1307–1315
( p < 0.01) percentage of kidding than nulliparous females in both experimental periods (1 and 6 months of cryopreservation). Finally, the prolificacy was also higher ( p < 0.05) in multiparous than in nulliparous goats. 4. Discussion This study defines, for the first time, the in vitro quality of caprine semen after a long time (1 year) of cryopreservation using a 152 8C ultrafreezer. In addition, the results showed that long-term storage of the semen did not alter the semen quality. Finally, fertility trials confirmed that semen straws, frozen and stored in ultrafreezers at 152 8C, remained fertile after 6 months of cryopreservation. Fresh semen quality was very similar between males, and the only observed differences were in ejaculate volume and sperm concentration. After thawing, the sperm motility (total and progressive) showed similar mean values between both freezing protocols, comparable to that obtained in frozen-thawed semen of other caprine breeds [2,11,22,25–27]. Most previous studies have included the use of liquid nitrogen to freeze the semen straws [2,11,22] and a few studies have frozen the semen samples without liquid nitrogen [1,14]; however, all of these studies required liquid nitrogen for subsequent storage. A preliminary work [20] developed with a low number of animals proposed the use of ultralow freezers, where liquid nitrogen was not necessary for semen cryopreservation. In that study [20], the thawed semen showed mean values of progressive motility ranging between 31% and 42% (assessed subjectively) as compared to the values of 29–45% (assessed by CASA system), obtained in the present study. It seems evident that the ULF did not modify the parameters of sperm motility after freezing, showing highly correlated mean values between both protocols. The sperm motility was not modified throughout the experimental period in any individual buck, showing reasonably constant mean values for 1 year after freezing. These findings are in agreement with other published data reporting no variation of the seminal quality throughout the period of cryopreservation [28]; however, other studies have found deterioration in the viability of spermatozoa after long-term frozen storage [29,30]. On the other hand, the results of our study detected differences between bucks in the ability of the semen to remain viable after freezing. This individual variability in the sperm motility after freezing is relatively independent of fresh semen quality and it seems necessary to assess the seminal quality after
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freezing-thawing to define the donors bucks as ‘‘good freezers’’ or ‘‘bad freezers’’ [1]. The percentage of acrosome membrane integrity in both freezing protocols showed values (mean) around 60–70%. In other caprine breeds, after freezing and storing with LN, the acrosome membrane-intact spermatozoa ranged between 40% and 70% [11,12,22]. In addition, in semen samples assessed after cryopreservation with ULF, the acrosome integrity reached a mean value around 55% [20]. With regard to the percentage of abnormal sperm cells post-thawing, both freezing protocols (LN and ULF) showed no significant differences; in addition, in any individual male, this spermatic feature was similar over the test period and no significant differences were found when bucks were compared themselves. Different studies have assessed the percentage of normal sperm cells post-thawing and found a range between 50% and 80% after freezing with LN [2,9,11,12]; no results for abnormal sperm cells after freezing with the ULF protocol have been reported until present. According to our results, the ULF did not generate a higher percentage of sperm abnormalities than other freezing protocols. The kidding rates observed in our study (43.2% and 39.2% for LN and ULF, respectively) were similar to the fertility results in other caprine breeds after cervical insemination with frozen-thawed semen: 39% in the Cashmere goat [7], and 39–42% in Saanen goats [14]. Other studies reached higher fertility rates after artificial insemination with frozen-thawed semen: 47.6% in the Florida breed [12], 51% in Angora goat [30], 57% in the Murciano-Granadina goats [9] and 57–61% in Saanen and Alpine goats [1]. However, most of these studies were carried out with multiparous goats and our results showed a remarkable influence of the number of previous parities on the fertility rates. Nulliparous goats showed a significantly lower kidding rate (28–33%) than the multiparous goats (45–50%). Dorado et al. [12] found a higher fertility rate in multiparous (58.8%) than in uniparous goats (0%) after artificial insemination with semen frozen-thawed; in addition, it has been shown that after hormonal induction of estrus, nulliparous goat were less fertile than multiparous goats when inseminated intracervically [1]. The lower fertility rates observed in nulliparous goats could be explained by the time of insemination relative to ovulation; in multiparous goats, the insemination took place around the middle of the estrous period (estrus length: 41.2 h), while in nulliparous goats, the inseminations were carried out in the second stage of estrus (estrus length: 32.0 h). A higher fertility rate has been demonstrated in goats inseminated in the first stage of the estrus period [1]. On the other
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hand, it should be noted that the nulliparous goats showed higher degree of nervousness and the stress involved in the insemination technique could have generated the lower fertility rate observed in our study. Different studies have assessed different factors affecting fertility rates after artificial insemination with frozen-thawed semen [1,9,12]. It has been found that the fertility was better when the semen was collected in the breeding season, rather than in the non-breeding season [31]; in our study, semen collection was carried out during the breeding season. Consequently, the seasonal influence on sperm freezability and, thus, on fertility should be ruled out. In addition, the semen was pooled before freezing to avoid the influence for differences between bucks in the ability of the semen to remain viable following freezing [1]; moreover, the semen straws were assessed before insemination and only semen straws with a minimum requirement of progressive motility were used for AI. On the other hand, the influence of the technician could be ruled out since both inseminators had the same experience and no differences in fertility were detected between technicians. Finally, different studies have established the relationship between the site of semen deposition and the fertility rates [5,6,31], showing a higher percentage of fertility rate when the semen is deposited into the cervix or into the uterus than if the AI was vaginal. In our study, most of the inseminations (90%) were performed into the cervix. Few studies that have reported fertility rates after AI with frozen-thawed semen have valued the influence of the storage time of the semen straws on the fertility. Fougner [32] obtained a 63.4% kidding rate after intracervical inseminations with semen stored in LN for 1–3 years and no significant differences were found in fertility rates throughout the experimental period. However, other study has detected a decline in the seminal quality (in vitro) after storing the semen for 6 months in LN [6]. In our study, no differences were observed in the kidding rate after artificial insemination with semen frozen-thawed after 1 or 6 months of storage. Therefore, these results confirmed that the storage time of frozen semen had no influence on the subsequent fertility. Before this study, only one work [20] had proposed the use of the ULF to freeze and store caprine semen, but that study was carried out for a short storage time (2 months) and no fertility trials were performed. In our study, no differences were observed between both freezing techniques, presenting similar results of sperm quality in vitro throughout the experimental period (1 year). In addition, the fertility results confirmed that caprine semen remained fertile after cryopreservation
with the ULF, showing a kidding rate and prolificacy practically equal to that observed after AI with semen frozen with LN. Ultra-low temperature freezers have advantages over freezing with liquid nitrogen. It has been reported [18,19] that the higher storage capacity of the ULF, the lower time elapsed to freeze the straws after equilibration and the sample handling is easier than with LN; in addition, unlike cryopreservation with liquid nitrogen, periodic reposition is not required, and the global cost of the freezing protocol is cheaper. However, an important disadvantage of the ULF protocol is the limited mobility of the freezer that could reduce the efficacy of the AI in farms far from the lab. To avoid this limitation, at present, new research lines are developing, where semen straws frozen and stored in the ULF will be moved to liquid nitrogen to subsequently test the semen quality (in vitro and in vivo). In addition, further studies are needed to assess the fertility of caprine semen frozen using ULF after storing the semen for several years. References [1] Leboeuf B, Restall B, Salamon S. Production and storage of goat semen for artificial insemination. Anim Reprod Sci 2000;62:113–41. [2] Hidalgo M, Rodrı´guez I, Dorado JM. The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability. Anim Reprod Sci 2007;100:61–72. [3] Roca J, Carrizosa JA, Campos I, Lafuente A, Va´zquez JM, Martı´nez E. Viability and fertility of unwashed Murciano-Granadina goat spermatozoa diluted in Tris-egg yolk extender and stored at 5 8C. Small Ruminant Res 1997;25:147–53. [4] Leboeuf B, Forgerit Y, Bernelas D, Pougnard JL, Senty E, Driancourt MA. Efficacy of two types of vaginal sponges to control onset of oestrus, time of preovulatory LH peak and kidding rate in goats inseminated with variable numbers of spermatozoa. Theriogenology 2003;60:1371–8. [5] Paulenz H, So¨derquist L, Adnoy T, Soltun K, Saether PA, Fjellsoy KR, et al. Effect of cervical and vaginal insemination with liquid semen stored at room temperature on fertility of goats. Anim Reprod Sci 2005;86:109–17. [6] Ritar AJ, Salamon S. Fertility of fresh and frozen-thawed semen of the Angora goat. Aust J Biol Sci 1983;36:49–59. [7] Ritar AJ, Ball PD, O’May PJ. Artificial insemination of Cashmere goats: effects on fertility and fecundity of intravaginal treatment, method and time of insemination, semen freezing process, number of motile spermatozoa and age of females. Reprod Fertil Dev 1990;2:377–84. [8] Karatzas G, Karagiannidis A, Varsakeli S, Brikas P. Fertility of fresh and frozen-thawed goat semen during the nonbreeding season. Theriogenology 1997;48:1049–59. [9] Salvador I, Viudes-de-Castro MP, Bernacer J, Go´mez EA, Silvestre MA. Factors affecting pregnancy rate in artificial insemination with frozen semen during non-breeding season in Murciano-Granadina goats: a field assay. Reprod Domest Anim 2005;40:526–9.
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