www.journals.elsevierhealth.com/periodicals/the

Theriogenology 66 (2006) 974–981

Effect of solid storage on caprine semen conservation at 5 8CI. Salvador a, J. Yaniz b, M.P. Viudes-de-Castro a, E.A. Gomez a, M.A. Silvestre a,b,*

a Centro de Investigacion y Tecnologıa Animal, Instituto Valenciano de Investigaciones Agrarias (CITA-IVIA), Moncada, Valencia, Spainb Department of Animal Production, University of Zaragoza, Huesca, Spain

Received 1 August 2005; received in revised form 20 February 2006; accepted 26 February 2006

Abstract

In this work, we investigated the effect of storage in solid-phase extender on buck semen conserved at 5 8C. Furthermore, we

studied the effect of addition of cysteine to the extender and the washing of seminal plasma on sperm survival. In Experiment 1,

milk-based extender (M) was used as a control to study the effect of solid media storage (G) and cysteine supplementation (C), and

the combination of both (GC), on in vitro sperm survival of washed and non-washed semen, conserved up to 72 h at 5 8C. Motility,

acrosome integrity (NAR) and hypo-osmotic swelling tests (HOST) were evaluated to assess in vitro sperm survival. In Experiment

2, an artificial insemination (AI) field trial was performed to compare G versus M. Solid media (G) maintained motility of

spermatozoa during storage higher than any other extender (67% G versus 62% GC; 61% M and 59% C; P < 0.05), but there was no

difference in NAR or HOST between extenders (P > 0.05). No improvement in sperm viability was obtained by addition of cysteine

to the media. Washing of semen improved motility (65% versus 60%; P < 0.05), NAR (70% versus 64%; P < 0.05) and HOST

(37% versus 28%; P < 0.05). No significant differences in fertility were obtained between G and M extenders (47% versus 41%;

P > 0.05). In conclusion, washing of semen and dilution in gelatin-supplemented milk extender (solid storage) appears to be a

successful method for goat semen storage at 5 8C.

# 2006 Elsevier Inc. All rights reserved.

Keywords: Goat; Chilled semen; Extender; Gelatin; Cysteine

1. Introduction

Cooled semen suffers from a decrease in motility and

structural integrity, accompanied by a decline of the

survival in the female reproductive tract, reduction in

fertility and increased embryonic loss [1]. However, in

general, this damage is lower than in frozen-thawed

semen. Fertility using semen stored at room temperature

or cooled at 5 8C is higher than when frozen semen is

used [2–6]. On the other hand, artificial insemination

(AI) plays an important role in milk goat breeding,

improving the connexion among farms, allowing a

* Corresponding author. Tel.: +34 96 3424000;

fax: +34 96 3424001.

E-mail address: masilver@ivia.es (M.A. Silvestre).

0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2006.02.042

higher accuracy in the evaluation of males and for

diffusion of animals with the highest genetic values. A

correct progeny test requires a great number of

inseminations, but this number could be reduced when

fertility increases. So, using chilled semen, the number

of inseminations required to test the future males could

be reduced. However, nowadays, fresh or cooled semen

have to be inseminated in a short period of time (1–12 h)

otherwise fertility is reduced [7]; so the use of cooled

semen is feasible only when most of the animals live in

a small geographical area. It would be of great interest,

from the breeding program point of view, to develop

protocols for goat semen conservation aimed to prolong

the potential fertility of cooled semen, as occurs in boar

semen.

Certain enzymes in the seminal plasma, originating

from the bulbourethral gland secretion, catalyse the

I. Salvador et al. / Theriogenology 66 (2006) 974–981 975

hydrolysation of egg yolk lecithin and milk triglycer-

ides of the extender, releasing sperm-toxic products

(lysolecithin and fatty acids) that lead to subsequent

sperm deterioration [8–11]. Removal of seminal plasma

by washing buck semen has been reported to increase

the percentage of live spermatozoa and their motility

during storage in egg yolk or milk diluents, improving

their in vitro capacity to withstand freeze-thawing in

both types of extenders [12,13]. However, in Murciano-

Granadina goat, Roca et al. [3] did not obtain any

improved effect of washing seminal plasma on in vitro

viability or in vivo fertility of chilled semen diluted in

Tris extender. Discrepancies about the effect of washing

seminal plasma in semen conservation exist in the

literature, which could be due to differences between

breeds, season, buck or diluent [3,9,13–15].

On the other hand, during storage in liquid phase,

sedimentation of cells normally occurs and, in

consequence, great pH fluctuations and increases in

toxic metabolic products may occur in this region [16].

As solid medium prevents sedimentation, the use of

gelatin could be an appropriate approach to overcome

this problem and to improve liquid semen storage. The

use of gelatin has been assayed in rabbit [16] and in ram

[17], with positive effect on in vitro viability and

integrity of spermatozoa during storage. In rabbit, solid

storage in gelatin-supplemented extender maintained

sperm fertility potential for 72 h [18].

Another method of avoiding the effect of toxic

metabolic products is the addition of antioxidants.

Spontaneous lipid peroxidation is associated with loss

of motility and decline of fertility of mammalian

spermatozoa [19–21]. It has been demonstrated that the

addition of antioxidants (catalase, superoxide dismu-

tase, cytochrome C, and glutathione peroxidase) can

improve the survival time of buck spermatozoa when

stored in a liquid form, but this was not reflected in

better in vitro fertilisation rates [22]. There appear to be

very few works involving liquid storage of buck semen

in the presence of antioxidants. In a recent study in pigs,

Funahashi and Sano [23] found that viability and

penetrability of spermatozoa in vitro were significantly

improved by adding 5 mM cysteine, a precursor of

intracellular glutathione biosynthesis, to preservation

solution. To our knowledge, the use of cysteine as

antioxidant in goat semen preservation has not been

reported.

Therefore, the aim of the present work was to study

the effect of storage in solid-phase extender at 5 8C on

buck semen viability and fertility. Moreover, we studied

the effect of addition of cysteine to the extender and

washing of seminal plasma on in vitro sperm survival.

2. Materials and methods

2.1. Animals and semen collection

Semen collection was performed following Silvestre

et al. [24]. Briefly, semen was collected by artificial

vagina from seven 12–24-month-old Murciano-Grana-

dina bucks, under uniform nutritional conditions.

Immediately after collection, the ejaculates were

immersed in a warm water bath at 30 8C until their

assessment in the laboratory. Semen assessment was

performed in approximately 20 min.

2.2. Extenders

In the Experiment 1, four extenders were tested:

Milk (M): milk-based extender was composed of

long-life ultra-heat-treated (UHT) skimmed-milk

and prepared following Corteel [12] protocol.

Briefly, 10 g of skimmed-milk powder was diluted

in 100 ml water containing 0.2 g D-glucose, heated in

a water bath at 92 8C for 10 min and cooled at room

temperature.

Gel (G): gel extender was prepared by addition of

1.5% (v/v) gelatin type A, gel strength 2408 Bloom,

to M extender.

Cysteine (C): this extender was prepared by supple-

mentation of the M extender with 5 mM cysteine.

Gel + cysteine (GC): this extender was prepared by

supplementation of the G extender with 5 mM

cysteine.

Penicillin and streptomycin were added to all

extenders, in order to prevent microorganism contam-

ination (50 mg of streptomycin and 50,000 UI of

penicillin per 100 ml of media). All chemicals were

obtained from Sigma Co. (St. Louis, MO, USA) unless

otherwise stated.

2.3. Semen preparation

Volume of ejaculates was measured in a conical tube

graduated at 0.1 ml intervals, and concentration was

determined by a spectrophotometer (Acucell, IMV,

France) calibrated for goat species (1:400 dilution rate).

Only those ejaculates surpassing motility values of 75%

(evaluation described below) and sperm concentration

of 3 � 109 spermatozoa/ml, were mixed in a pool

balancing the sperm contribution of each male [1].

The pool of semen was split into two equal volumes,

one of them to be removed from its seminal plasma by

I. Salvador et al. / Theriogenology 66 (2006) 974–981976

washing and the other one maintaining its seminal

plasma. Semen was washed by centrifugation 10 min at

900 � g following dilution 1:10 (semen/Tris, v/v). Final

concentration of Tris in diluted semen for washing was

250 mM [25]. After centrifugation, supernatant was

gently removed avoiding, as far as possible, loss of

spermatozoa.

After washing procedure, both pools (washed and

non-washed) were diluted with the above described

extenders to a concentration of 600 � 106 spermatozoa/

ml, packaged in 0.25 ml straws and stored at 5 8C.

Cooling rate from room temperature to 5 8C was 0.2–

0.3 8C/min in a freezer unit.

2.4. Evaluation of sperm samples

2.4.1. Motility maintenance test

In Experiment 1, thermal resistance test (TRT) was

performed as follows: semen samples were light-

washed by centrifugation in Tris (5 min at 600 � g)

followed by re-dilution in Tris (1:4) and incubation at

37 8C in a water bath [26]. One sample from each

treatment at 0, 5, 24, 48 and 72 h of storage at 5 8C was

evaluated after 0, 2, 4, 6 h of incubation to determine

sperm motility. Sperm motility was assessed diluted in a

sodium citrate solution and under a phase-contrast

microscopy system (100� magnification) with a

warmed plate at 37 8C.

2.4.2. Acrosome integrity (NAR)

Samples were evaluated immediately after warm-

ing-up following 0, 5, 24, 48 and 72 h of storage at

5 8C. Percentage of spermatozoa population with

normal acrosome (NAR) was measured. As performed

in ram spermatozoa [27], we diluted (1/100) 10 ml of

semen sample in a saline solution (NaCl 0.9%) with

2% of glutaraldehyde, and percentage of spermatozoa

population with non-reacted and normal acrosome

was scored under phase-contrast microscopy (1000�magnification).

2.4.3. Osmotic resistance test (HOST)

Samples were evaluated immediately after warm-

ing-up following 0, 5, 24, 48 and 72 h of storage at

5 8C. We performed an osmotic resistance test used by

Paulenz et al. [1] in ram with some modifications: An

aliquot of semen (10 ml) in 100 ml of hypo-osmotic

solution (100 mOs mol/kg:1 g sodium citrate in

100 ml water) was incubated 1 h at 37 8C, and

percentage of spermatozoa population with swollen

tail was scored under phase-contrast microscopy

(400� magnification).

2.5. Artificial insemination fertility trial

2.5.1. Animals

This trial was conducted in the Mediterranean area of

Spain. The inseminations were conducted from

February to October. Before insemination, females

were oestrus synchronised by a hormonal treatment

(described below). Ultrasound scans of females were

performed before the synchronisation treatment appli-

cation, excluding those females diagnosed pregnant or

with pseudo-pregnancy. All the inseminated females

were multiparous or with at least one parity.

2.6. Insemination procedure

The oestrus synchronisation treatment was done

following Leboeuf et al. [4] and adapted to our

conditions [28]. Briefly, 11 � 1 days vaginal sponge

with 40 mg of progestagen fluorogestone acetate (FGA,

Sincropart, Ceva, Loudeac, France) and intramuscular

injection, of 2.5 mg of prostaglandin F2a (PGF2a,

Enzaprost, Ceva, Loudeac, France) and 300 IU of

PMSG (Sincropart, Ceva, Loudeac, France) 48 � 1 h

before the vaginal sponge removal.

Goats were inseminated once with 150 � 106 sperm

via cervix at 45–47 h after the removal of vaginal

sponges, using a speculum with an attached light source

and an ovine-caprine AI catheter (IMV Technologies,

L’Aigle, France). Semen was deposited in the genital

tract as deeply as possible without harming the cervix

epithelia.

2.7. Pregnancy assessment

Pregnancy rate was determined by trans-abdominal

ultrasound (5.0 MHz) six weeks (35–40 days) after

insemination.

2.8. Experimental design

In Experiment 1, four replicates of each experi-

mental group were performed in a complete 4 � 2 � 4

factorial design. Milk-based extender (M) was used as a

control to study the effect of solid media storage

(gelatin) and antioxidant (cysteine) supplementation to

the extender, in washed and non-washed semen

conserved up to 72 h (0, 24, 48, 72 h) at 5 8C. The

whole semen processing procedure is illustrated in

Fig. 1.

In Experiment 2, a field trial assay was performed in

148 goats using two types of semen: semen diluted in C

extender and semen diluted in M, as control. Semen was

I. Salvador et al. / Theriogenology 66 (2006) 974–981 977

Fig. 1. Pool (P) was split in two equal volumes, washed (W) and

non-washed (NW). After washing, both pools were split and diluted

in milk (M), gel (G), gel + cysteine (GC) and cysteine (C) based

extender, respectively. Semen was stored at 5 8C for 0, 24, 48, 72 h.

One straw from each treatment was evaluated for sperm quality

(motility; membrane integrity: HOST; acrosome: NAR). Motility

was further evaluated every hour of incubation (0, 2, 4, 6 h) at

37 8C.

Table 1

Significance based on GLM for the effects of extender, washing,

storage time, incubation and all the interactions on subjective motility

(MOT), acrosome integrity (NAR) and hyposmotic test (HOST)

Source of variation Sperm viability parametersa

MOT NAR HOST

Washing of semen *** *** ***

Extender *** NS NS

Storage time *** *** ***

Washing � extender NS NS NS

Extender � storage ** NS NS

Incubation ***

Extender � incubation NS

Storage � incubation **

Washing � incubation NS

NS: not significant.a NAR and HOST assessments were only performed at 0 h, only

motility was assessed in TRT (incubation).** P < 0.01.

*** P < 0.001.

Table 2

Least square means (SE) of percentage of subjective motility (MOT),

acrosome integrity (NAR) and hyposmotic test (HOST) for the

different factors studied

Source of variation Sperm viability parameters*

MOTa NAR HOST

Extender

Gel (G) 67 (0.73) a 69 (1.52) 34 (1.99)

Cysteine (C) 59 (0.73) c 67 (1.52) 32 (1.99)

Gel + cysteine (GC) 62 (0.73) b 65 (1.52) 31 (1.99)

Milk (M) caprine 61 (0.73) bc 68 (1.52) 34 (1.99)

Washing of semen

Washed 65 (0.52) a 70 (1.10) a 37 (1.40) a

Non-washed 60 (0.52) b 64 (1.10) b 28 (1.40) b

Storage (h)

0 73 (0.73) a 89 (1.52) a 48 (1.99) a

24 60 (0.73) b 69 (1.52) b 30 (1.99) b

48 60 (0.73) b 60 (1.52) c 31 (1.99) b

72 56 (0.73) c 50 (1.52) d 21 (1.99) c

a Incubation effect is included to estimate least square means in

motility, but not in NAR and HOST.* Least square means in columns and within each source of varia-

tion, without letters (a–d) in common, are significantly different

(P < 0.05).

washed to eliminate seminal plasma prior to dilution in

the respective extender and stored at 5 8C for 18–20 h

before AI.

2.9. Statistical analysis

In Experiment 1, analysis of variance of the data was

performed using a general linear model using Stat-

graphics Plus 4.1 Software (Manugistics Inc., Rock-

ville, MD). We included the effects of extender,

washing, storage time, incubation and all single

interactions in the model. Least square means were

used to perform a pairwise comparison of the different

extenders, washing of seminal plasma. The level of

significance was established at P < 0.05. In Experiment

2, a chi-square analysis was performed to analyse

fertility data.

3. Results

3.1. Experiment 1

Significance for the effects of extender, washing of

semen, storage time, incubation on motility, acrosome

integrity and hyposmotic test are shown in Table 1.

Washing of semen and storage affected all sperm

parameters (motility, NAR and HOST) evaluated

I. Salvador et al. / Theriogenology 66 (2006) 974–981978

Table 3

Pregnancy rate in goats inseminated with chilled (5 8C) semen diluted

and stored 18–20 h in two different phase milk-extenders: liquid or

solid

Extender Pregnancy rate (%)

(no. of does inseminated)

Liquid-phase 41 (70)

Solid-phase 47 (78)

No difference in pregnancy rate was obtained between extenders

(P > 0.05).

(P < 0.001), while extender only affected motility

(P < 0.001). Motility was affected (P < 0.001) by

incubation during TRT.

Differences within the sperm parameters between

extenders, storage time and washing of semen are

indicated in Table 2. Semen stored in solid conditions

(G) without supplementation of cysteine showed the

highest motility (P < 0.05) in comparison with the

other groups. No difference was obtained in NAR and

HOST in any supplemented extender in comparison

with the control extender. Sperm motility was less

depressed with time of storage in G extender compared

with the other experimental extenders, although this

effect was not observed in NAR or in HOST (Fig. 2).

Washing of semen improved all the semen parameters

evaluated (P < 0.05; Fig. 3). Semen stored for 72 h

showed earlier depression in sperm motility during TRT

(Fig. 4).

Fig. 2. Least square mean interval of percentage of subjective motile (motility

of fresh pooled semen from seven bucks, diluted in four different extenders –

of storage at 5 8C.

3.2. Experiment 2

The pregnancy rates after cervical insemination

stored 18–20 h washed spermatozoa diluted in M and G

extender are presented in Table 3. No significant

difference was observed between does inseminated with

semen extended and stored in liquid or solid-phase.

), normal acrosoma (NAR) and osmotic resistant (HOST) spermatozoa

(&) G; (^) C; (~) GC; (�) M – and evaluated after 0, 24, 48 and 72 h

I. Salvador et al. / Theriogenology 66 (2006) 974–981 979

Fig. 3. Least square mean interval of percentage of subjective motile (motility), normal acrosoma (NAR) and osmotic resistant (HOST) spermatozoa

of fresh pooled semen from seven bucks, washed and no washed semen (&: W; ^: NW) and evaluated after 0, 24, 48 and 72 h of storage at 5 8C.

Fig. 4. Least square mean interval of percentage of subjective motile

(motility) spermatozoa after incubation at 37 8C during 6 h (TRT) of

fresh pooled semen from seven bucks stored 0, 24, 48 and 72 h at 5 8C.

Symbols are: (^) 0; (&) 24; (~) 48; (�) 72 h, respectively.

4. Discussion

In this work, we studied the effect of supplementa-

tion of the milk-based extender with gelatin, for solid-

phase storage, with cysteine, as antioxidant, and the

combination of both, on in vitro survival of sperma-

tozoa. In addition, we also studied the effect of washing

the seminal plasma. In goat semen, removal of seminal

plasma by washing the spermatozoa has been proved to

increase the percentage of live cells and their motility

during storage in milk diluents [12]. In the present

work, in accordance with the latter, motility and

membrane integrity (HOST) were improved when

semen was washed previously. Thus, removal of

seminal plasma could be necessary for successful

chilled conservation of buck semen when milk-based

extender is used.

Addition of gelatin to the extender improved motility

of goat spermatozoa during storage at 5 8C. In this way,

a positive effect on in vitro viability and integrity of

semen by the addition of gelatin has also been reported

I. Salvador et al. / Theriogenology 66 (2006) 974–981980

in rabbit [16] and in ram [17], as well as on in vivo

fertility in rabbit [18]. The manner in which gelatin

improves sperm viability remains unclear. When semen

is stored in liquid media for a certain time, live and dead

spermatozoa are deposited at bottom. Reactive oxygen

species are produced by dead spermatozoa in bull and in

ram semen, and play an important role in diluted sperm

survival [29,30]. At this region of sedimented cells, pH

may be lower and concentration of some toxic

metabolic products may be higher. Storage in solid

phase may exert a beneficial effect in two ways [17]: (1)

sedimentation of sperm cells is avoided and, conse-

quently, changes in composition of the medium are

reduced and (2) immobilisation of spermatozoa would

reduce metabolic demands of sperm motion, maintain-

ing their fertilisation potential [18].

Cooling processes produce physical and chemical

stress on the sperm membrane that reduces sperm

viability and their fertilising ability [31]. The cold

shock of spermatozoa is associated with oxidative

stress induced by reactive oxygen species generation

[32]. Supplementation with antioxidants has been

proved to improve the viability and motility of liquid

or cryo-preserved spermatozoa of several mammalian

species: goat [22], ram [33], bull [21] and boar

[23,31]. In our study, we investigated the antioxidant

effect of cysteine, alone or in combination with solid

medium. In our conditions, no improvement in sperm

viability was obtained by addition of cysteine to the

media. These results do not agree with results obtained

by Funahashi and Sano [23], who observed that the

supplementation of 5 mM of cysteine improved the

viability and functional integrity of boar spermatozoa

during liquid preservation at 10 8C for at least 14 days.

Thiol compounds such as cysteine and glutathione

have been shown to protect the motility and glycolytic

activity of spermatozoa in vitro against oxidizing

agents [34,35]. Glutathione peroxidase is found in a

number of cells and can reduce many types of reactive

oxygen species through the glutamyl cycle [36].

Moreover, glutathione (gamma-glutamyl-cysteinyl-

glycine) improves development of the male pronu-

cleus at fertilisation and has also been implicated in

promoting the development of preimplantation of goat

embryos [37], and the addition of 1 IU/ml of

glutathione peroxidase to Tris-glucose-egg yolk

extender has been reported to improve the motility

of goat spermatozoa during liquid storage at 5 8C up to

8 days [22]. Cysteine is a precursor of intracellular

glutathione biosynthesis, and thus, it was thought

cysteine might also increase viability of buck

spermatozoa. In spite of our results, we are not able

to conclude that addition of cysteine in diluted buck

semen is not beneficial for improving survival of buck

spermatozoa. It is possible that our results could

respond to an inappropriate concentration of cysteine

for buck semen, as concentration was directly applied

from that assayed in boar [23], or to a failure in any

other conditions necessary for the buck spermatozoa

to be able to recycle glutathione properly (time of

storage, temperature, coenzymes [38]). Further

research is required to study the beneficial effect of

antioxidants in goat semen.

In the artificial insemination trial performed to

compare fertility of chilled semen stored 16–18 h in

liquid-phase or in solid-phase, obtained pregnancy rates

(41% versus 47%, respectively) were no significantly

different. These results were lower than fertility results

published in this breed (70.3%) using cooled semen

(5 8C) and held for 6–36 h [3]. However, in this work

[3], authors did not indicate the percentage of goats

inseminated before and after 12 h post-semen collec-

tion. In other works [6] obtained a non-return-rate (25-

day NR) of 86.2% in Norwegian Dairy goats using

liquid semen stored at room temperature and insemi-

nated 3–8 h after collection. The lower results obtained

in this work could be explained by the fact that

prolonged time of storage of the chilled semen, beyond

12–24 h, is known to reduce the fertility [7]. In fact, in a

previous work (data not published), we obtained 74%

(35/47) of goats pregnant using fresh semen insemi-

nated 2–5 h after collection.

In conclusion, even though in this work significant

improvement in fertility with solid-phase storage of

semen was not reached, washing of semen and dilution

in gelatin-supplemented milk extender (solid storage)

appears to be a successful method for goat semen

storage at 5 8C. Larger field assays should be performed

to confirm these findings.

Acknowledgments

This work was supported by INIA (RTA03-100). I.

Salvador was supported by a research grant from

Conselleria de Agricultura, Pesca, y Alimentacion

(Generalitat Valenciana). The authors would like to

thank N. Macowan for revising the English version of

this manuscript.

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