Ž .
ing for 20.1, followed by abnormal acrosome structure 12.9 and the presence of Ž
. cytoplasmic droplets
11.1 . Most of the biochemical components of semen are comparable to those of other farm animals with the exception of small amounts of
Ž .
Ž .
fructose 4–6 mgrdl and citric acid 4.3 mgrdl which may be due to the absence of Ž
. vesicular glands Garnica et al., 1995 . There is no significant difference regarding the
Ž .
biochemical components between the 3- and 6-year-old alpaca males Table 3 . 6.2. Characteristics of camel semen
Semen of camels is of grey to milky white colour, and as in lamoids, has a viscous consistency immediately after collection. If it is allowed to stand for 10–15 min, it
partially liquefies and the spermatozoa attain motility. Semen used for AI or storage should have at least the characteristics shown in Table 4, as recommended also by
Ž .
Ž .
Ž .
Tingari et al. 1986 , Taha Ismail 1986 and Merkt et al. 1990 .
7. AI and storage of semen
7.1. Use of fresh undiluted and diluted semen in lamoids AI in lamoids has been impeded by difficulties in semen collection and semen
handling. Nonetheless, there is some progress and a brief discussion is given below. Ž
. The first attempt on AI Fernandez-Baca and Novoa, 1968 involved the use of 42
female alpacas that were inseminated with fresh undiluted semen from two vicunas and Ž
. four paco vicunas cross between alpaca and vicuna . Semen samples were obtained by
electroejaculation and the females were induced to ovulate by copulation with vasec- tomized males. Insemination was done immediately after induction of ovulation and the
semen was deposited at the point of bifurcation between the left and right uterine horns. A cria was born after 340 days of gestation.
The second study to determine the best time for insemination after induction of Ž
ovulation used 96 alpacas and fresh semen collected by electroejaculation Calderon et .
al., 1968 . The females were slaughtered 3 days after insemination and 75 had fertilised eggs when insemination was carried out between 35 and 45 h after induction of
ovulation, in contrast to 12.5 when females were inseminated immediately or 18 h after induction of ovulation. The fertilization rate decreased to 58.3 when insemination
occurred 52 h after induction of ovulation.
Ž .
In further study Leyva et al., 1977 , 83 alpacas and 11 llamas were inseminated with semen from a vicuna and four paco vicunas. The semen was collected by electroejacula-
tion and was inseminated undiluted through the cervix into the uterine horns. Ovulation was induced by administration of hCG and copulation with vasectomized males. In
addition, 10 IU oxytocin was administered to 24 females mated with vasectomized males. The fertility rate was 48 for females treated with hCG and 11 for females
mated with vasectomized males. Oxytocin had no effect.
Ž .
In a report by Bravo et al. 1997a , using semen collected by AV, 20 alpacas were inseminated via the cervix with undiluted semen and 20 alpacas by laparoscopy into the
uterine horns with diluted semen. A mean fertility rate of 60 was obtained with no Ž
. differences in fertility for methods of semen deposition. Pacheco 1996 used semen
Ž .
Ž .
collected by AV and diluted with egg yolk 10 –citrate 3 for insemination of 80 alpacas. The semen was deposited into the uterine horns by cervix fixation through the
rectum and ovulation was induced by treatment with GnRH 24 h before insemination. The fertility rate was 60.
Ž .
In a study by Quispe 1996 , 8 million motile spermatozoa appeared to be sufficient Ž
. for AI in alpacas. In the report of De la Vega 1996 , semen collected by AV was
Ž .
Ž .
diluted with phosphate-buffered saline 8 and mixed with cria alpaca serum 10 . The semen was deposited through the cervix at the bifurcation of the uterine horns. The
Ž .
pregnancy rate was 54r133 40 . The best fertility rate was obtained when insemina- tion took place 30 h after induction of ovulation with a vasectomized male. In an
Ž .
incubation test 358C for viability of spermatozoa, egg yolk–citrate proved to be more Ž
efficient than saline–phosphate or skim milk as diluting media for alpaca semen Bravo .
et al., 1997b . The methodologies and results of AI in lamoids are summarized in Table 5.
7.2. Frozen storage of semen in lamoids There are few reports on freezing lamoid semen. In the report of McEvoy et al.
Ž .
1992 , llama semen collected by electroejaculation was frozen in straws after one-step Ž
. dilution 1:2 with Tris–egg yolk–glycerol diluent. Only 10 of spermatozoa showed
Ž .
motility after thawing. On the other hand, Graham et al. 1978 observed 50, 45 and 30 motile spermatozoa before freezing, after thawing, and after 8 days of frozen
storage, respectively. Ž
. A further report on freezing lamoid semen is by Bravo et al. 1996b . Semen from
Ž three alpacas and two llamas was collected by AV and liquefied with collagenase 1
. mgrml to facilitate semen handling. For two-step dilation of semen sodium, citrate
Ž .
Ž .
2.9 –egg yolk 10 diluent was used. The second diluent fraction containing 7 glycerol was added at 15-min intervals in three equal parts when the semen diluted in
the first step was undergoing cooling to 48C in a refrigerator. The cooled semen was loaded into 0.25 ml straws and equilibrated for 2 h. After freezing in liquid nitrogen
vapor, the straws were plunged into liquid nitrogen. At the time of semen collection, 80 of spermatozoa showed oscillatory motility, and after final dilution and cooling
Ž
. before freezing , 60 of spermatozoa was progressively motile. The semen was kept
for 9 months before use. After thawing in water bath at 358C for 8 s, 30–40 of sperm cells had oscillatory movement. The frozen–thawed semen was used for the insemina-
tion of 19 female alpacas of which five were diagnosed pregnant 5 months later by ultrasonography, and gave birth to three male and two female crias.
7.3. Use of fresh and liquid-stored camel semen For fresh use and liquid storage of dromedary semen, a number of extenders have
Ž .
been used Sieme et al., 1990 . The best results to date have been achieved with three
P.W. Bra
Õ o
et al.
r Animal
Reproduction
Science 62
2000 173
– 193
185 Table 5
Different methodologies and results of artificial insemination in lamoids Author
Type of Collection
Ovulation Type of
Females Place of semen
Fertility Ž .
species method
induction semen
inseminated deposition
Ž .
Fernandez-Baca and Vicuna and Electroejaculation Teaser male
Undiluted 42 alpacas
Uterine horns via cervix one cria born
Novoa, 1968 paco vicuna
Ž .
Calderon et al., 1968 Alpaca Electroejaculation Teaser male and hCG Undiluted
96 alpacas Uterine body via cervix
75 after 35–45 h Ž
. Leyva et al., 1977
Vicuna and Electroejaculation Teaser and hCG
Undiluted 83 alpacas 11 llamas Uterine body via cervix
38 paco vicuna
Ž .
Quispe, 1996 Alpaca
AV hCG
Diluted, 80 alpacas
Deep uterus via cervix 57
egg yolk–citrate Ž
. De la Vega, 1996
Alpaca AV
Teaser male Diluted, PBS
133 alpacas Uterine body via cervix
40
a
Ž .
Bravo et al., 1996a,b Alpaca, llama AV hCG
Diluted, 19 alpacas
Deep uterus via cervix 26
egg yolk–citrate Bravo et al., 1997a
Alpaca AV
hCG Undiluted
40 alpacas Deep uterus
67 Ž
. via cervix and laparoscopy
a
Frozen–thawed semen.
Ž .
Ž .
commercial extenders: Green buffer IMV, L’Aigle, France , with 20 v:v egg yolk Ž
. Ž
. added, Laiciphos IMV and Androhep Waberski et al., 1989 , or a diluent containing
Ž .
Ž .
Ž .
11 w:v lactose and 20 v:v egg yolk Anouassi et al., 1992 , as compared with Ž
. Ž
other extenders such as skimmed milk Kenny et al., 1975 or glucose-EDTA Martin .
and Klug, 1979 . As the semen collection procedure can be rather prolonged, it may be advantageous to add about 1–2 ml of extender into the collection vessel before
collection. In general, the ratio of diluent to semen should be 3:1 with a diluent temperature of 30–358C when it is added slowly to the semen. Further work is required
to determine the optimum number of spermatozoa per insemination in the dromedary camel, but initial studies would indicate that at least a minimum of 100 = 10
6
motile Ž
. spermatozoa are needed
Anouassi et al., 1992 and fertility can be improved if
6
Ž .
300 = 10 motile spermatozoa are inseminated J.A. Skidmore, unpublished data . The diluted semen can be stored in a refrigerator at 48C for 24–36 h or in an
Ž .
equitainer Hamilton Thorn, Canvers, MA, USA , where the cans used to cool the equitainer are placed in a freezer for at least 24 h before use. The semen is sealed in a
plastic universal which is wrapped in two thermal ballast bags at room temperature and placed within a plastic cup inside the equitainer before closing the lid. The semen is
used for insemination 24 h later, provided it contains at least 35–40 motile spermato- zoa. While pregnancy rates of 50–60 have been reported in camels inseminated with
Ž .
fresh diluted semen within 30 min of collection Anouassi et al., 1992 , the conception rate decreased to 25–30 in camels inseminated with semen cooled–stored for 24 h. All
the pregnancies were achieved with cooled semen diluted in Green buffer q 20 egg Ž
. yolk J.A. Skidmore, unpublished data .
7.4. Frozen storage of camel semen Ž
. Split samples of semen were used by Seime et al. 1990 to compare different
freezing methods by assessing post-thaw morphology, motility and viability of sperma- tozoa in 1 sodium chloride solution at 388C. They found that the best freezing method
for dromedary semen was a modification of the technique developed for boar semen by Ž
. Westendorf et al. 1975 . Details of the method used are shown below.
Ž .
Ž .
Ž .
Ø Dilution of semen at 25–308C 1:1, vrv with lactose 11 –egg yolk 20 cooling
diluent. Ø
Cooling to 158C in 2.5 h.
6
Ž Ø
Second dilution to sperm concentration of 150 = 10 rml approximately 2r3 of total .
volume with cooling diluent. Ø
Cooling to 58C in 1.5 h.
6
Ž Ø
Further dilution to sperm concentration of 100 = 10 rml approximately 1r2 of total .
volume with the above lactose–yolk diluent containing 6 glycerol and 1.5 OEP Ž
. Equex ; final glycerol concentration approximately 2.
Ž .
Ø Filling of 4 ml straws Fa. Makrotub, Landshut, Germany with semen diluted to
final rate.
Freezing of straws: Ø
from 58C to y1208C by suspending in liquid nitrogen vapour for 20 min; Ø
from y1208C to y1968C by plunging into liquid nitrogen. Attempts to freeze dromedary semen using a combination of both Green and Clear
Ž .
Ž .
buffers IMV have been carried out by Skidmore et al. unpublished adopting the following protocol:
Ž .
Ø Dilution of semen at 25–308C 1:1, vrv with Green buffer containing 20 egg
yolk. Ø
Cooling to 48C in 2 h. Ø
Second dilution by addition of equal volume of Clear buffer containing 20 egg yolk and 11 glycerol; final glycerol concentration approximately 3.0.
Ø Equilibration at 48C for 30 min.
Ø Filling of 0.25 ml straws with semen diluted to final rate.
Ø Freezing of straws:
from 48C to y1408C by suspending in liquid nitrogen vapour for 20 min; from y1408C to y1968C by plunging into liquid nitrogen.
For freezing Bactrian semen, different glucose-, sucrose- and lactose-based diluents Ž
. were examined by Chinese workers Zhao, 1995; Zhao et al., 1990, 1996b .
In a comparison of different diluents using split semen samples frozen in ampoules, Ž
. the SYG-2 extender 73 ml of 12 sucrose–20 ml egg yolk–7 ml glycerol gave the
best results regarding post-thaw motility, survival and acrosome integrity of Bactrian Ž
. spermatozoa Zhao et al., 1996b .
The SYG-2 medium is used for two-step dilution by the following procedure: Ž
. Ø
First dilution 1:3 at 35–378C with non-glycerolated diluent. Ø
Holding the diluted semen at 208C for 2 h. Ø
Cooling and holding at 48C for 4 h. Ž
. Ž
. Ø
Addition 1:1 of glycerolated 7 diluent. Ø
Holding at 48C for 10 min. Ø
Transfer of diluted semen in 2 ml ampoules. Ø
Suspension of ampoules in liquid nitrogen vapour for 10 min. Ø
Plunging the ampoules into liquid nitrogen.
7.5. Thawing of camel semen Ž
. Thawing is best carried out in a water bath: small 0.25 ml straws at 408C for 10 s,
Ž .
large 4 ml straws at 508C for 40 s and ampoules at 50–558C for 1–2 min. The quality
Ž .
of thawed dromedary semen was improved by freezing in small straws 0.25 ml , but the use of larger straws was recommended to ensure deposition of an adequate amount of
semen in the uterus, which is necessary for inducing ovulation. However, this necessity has been overcome these days by treatment of camels with 3000 IU hCG or 20 mg
Buserelin 24 h before the insemination.
7.6. Fertility results for frozen–thawed camel semen Although remarkable fertility has been reported for Bactrians inseminated with
frozen–thawed semen, such results have not yet been obtained for dromedaries. Post-thaw motility has looked promising, but the pregnancy results to date do not reflect this.
The pregnancy rates obtained with frozen–thawed Bactrian semen after different ‘‘systems’’ of insemination are shown in Table 6. It should be noted that the mean
pregnancy rate of 95 after insemination with frozen–thawed semen was higher than the pregnancy rates of 60–65 obtained over 8 years after natural mating. It should also
be mentioned that by supplementation of semen with GnRH, it was possible to use the
Ž .
semen of one male for 450 inseminations Zhao, 1994; Zhao et al., 1996a . 7.7. Methods and timing of insemination
Ž Female lamoids that are receptive to the male assume a prone position ventral
. recumbency after a short period of chasing by the male, or adopt a copulatory position
next to mating alpacas. For the purpose of AI, all sexually receptive females are mated
Table 6 Ž
Pregnancy rates in Bactrian camel after AI with frozen–thawed semen semen frozen with SYG-2 diluent; .
Zhao et al., 1996a
a
Insemination system Number of
Number of Number of females
females females
pregnant at days Ž .
inseminated ovulated
60–90
b
Ž .
Ž .
Double AI 4q4 ml at 24-h interval 71
68 68 95.8
Ž .
Single AI 24 h after 1000 IU hCG injection 10
10 10 100
Ž .
Single AI 24 h after 200 IU LH injection 10
10 10 100
Ž .
Ž .
Single AI with double dose 8 ml 10
10 10 100
Ž .
Ž .
Single AI with single dose 4 ml 5
4 4 80
Ž .
Single AI with semen diluted 10-fold 45
45 45 100
c
q200 mg GnRHrdose Ž
. Single AI with semen diluted 15-fold
149 149
138 93
d
q200 mg GnRHrdose Ž
. Totals and mean
300 287
285 95
a
Ž .
Dose of inseminate: 4 ml two 2.0 ml ampoules .
b
Total number of motile sperm inseminated: 1.48=10
9
.
c
Total number of motile sperm inseminated: 290=10
6
.
d
Total number of motile sperm inseminated: 150=10
6
.
to a vasectomized male or the females are checked for follicular size by ultrasonogra- phy. A second alternative to induce ovulation is the administration of 750 IU hCG or
80–800 mg GnRH. So far, the best time for insemination is 36 h after induction of ovulation. In fact, 75 of females had fertilised eggs when slaughtered 3 days after
insemination in contrast to 40 when insemination was done at the time of induction of
Ž .
Ž .
ovulation Calderon et al., 1968 . This report has been confirmed by Quispe 1996 who obtained 60 pregnancy rate by inseminating 36 h after induction of ovulation.
Insemination is carried out with the female in standing position. Preferably, two people restrain the animal and the operator andror inseminator empties the rectum from fecal
material. The perineum is cleaned and the cervix held through the rectum while an insemination pipette loaded with semen is inserted into the uterine horns for deposition
of semen.
Oestrous female camels can be detected by teaser males. However, more accurate records can be obtained by rectal palpation combined with teasing or by ultrasonography
of the uterus and ovaries. When using ultrasonography or rectal palpation, the ovarian follicles should be noted and not be smaller than 12 mm in diameter. Insemination can
Ž .
be carried out with the female in a standing position dromedary or restrained in a Ž
. sitting position dromedary and Bactrian . The perineal region should be cleaned and the
semen deposited into the cranial part of the cervix or the body of the uterus. This can be achieved by using an insemination gun passed through the vagina and cervix with one
hand in the rectum holding the cervix, similar to the technique used in cattle. An alternative method is to use a porcine insemination catheter together with a tubular
speculum. The screw-like end of the catheter facilitates its passage through the annular rings of the cervix in the female camel.
In the dromedary camel, ovulation occurs between 36 and 48 h after GnRH of hCG injection and the optimum time for insemination with either fresh, liquid-stored or
frozen–thawed semen is 24 h after treatment. Good results have been obtained with fresh semen when insemination occurred 24 h after mating with a vasectomised male
Ž .
Anouassi et al., 1992 . In the Bactrian camel, ovulation occurs 30–48 h after mating or treatment with
hormones and the appropriate time for insemination with fresh semen is the first day of sexual receptivity. When frozen–thawed semen is used, it is advisable to inseminate
Ž .
twice at 24-h interval. Chen et al. 1985 reported that the incidence of ovulation was 87 after deep vaginal and 100 after uterine insemination. There is no information
regarding the optimum number of spermatozoa per insemination. All Bactrian females
6 y1
Ž .
ovulated after insemination with sperm numbers of 5–8 = 10 ml
Xu et al., 1985 and pregnancy rates of 50 have been obtained in dromedary camels with 100 = 10
6
Ž .
motile spermatozoa Anouassi et al., 1992 .
8. Conclusions
