SEMEN COLLECTION, EVALUATION, ARTIFICIAL INSEMINATION and CORRELATION of SEMEN PARAMETERS with PREGNANCIES in ALPACAS a Capsto

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SEMEN COLLECTION, EVALUATION, ARTIFICIAL INSEMINATION AND CORRELATION OF SEMEN PARAMETERS WITH PREGNANCIES IN ALPACAS

A Capstone Experience Manuscript

Presented by

Petra Szymkowicz

Completion Date: May 2012

Approved By:

Stephen R. Purdy, DVM, Veterinary and Animal Science Department, Chairperson

Carlos Gradil, DVM, Veterinary and Animal Science Department, Committee Member

ABSTRACT

Title: “Semen Collection, Evaluation, Artificial Insemination and Correlation of Semen Parameters with Pregnancies in Alpacas” Author: Petra Szymkowicz, Animal Science CE Type: Independent Capstone Thesis Approved By: Dr. Stephen R. Purdy, Veterinary and Animal Science Department Approved By: Dr. Carlos Gradil, Veterinary and Animal Science Department

The purposes of this study are to present information regarding semen characteristics and the potential for artificial insemination in 20 male alpacas and examine the correlation for these parameters with pregnancy rates. The samples were collected from an artificial vagina mounted in a breeding phantom and also from the cranial vagina of bred females immediately post copulation. 12 of the 20 males were evaluated 4 or more times using the breeding phantom and 24 postcoital breeding samples were evaluated. The postcoital samples were correlated with pregnancy results in the females in the study as determined by transrectal ultrasound. Male reproductive organs were initially examined by palpation of the testes, and ultrasound of the testes, bulbourethral glands, and prostate gland. Measurements and ultrasound pictures were recorded. Semen parameters evaluated for the samples included volume (phantom breedings only), sperm activity immediately after semen collection, live sperm percentage and sperm morphology from stained slides, and estimated sperm concentration immediately after semen collection (estimated as low, medium, or high). Data is presented on all measured parameters along with pictures. Ability to collect males using the breeding dummy was inconsistent. Of 101 attempted mounts of the phantom, 38% produced collections with a volume greater than 0.1 mL with sperm seen on the slides. Of the 45 collections in which activity percentages were recorded, 33% of these collections were successful with activity percentages greater than 0. A wide variation in semen parameters was found in individual males and among the males collected making the artificial insemination an unlikely prospect in alpacas. Sperm activity, live percentage, normal morphology percentage, and estimated concentration were evaluated with respect to pregnancies achieved in the study females. No direct correlation for any of these parameters was found in these males and females. INTRODUCTION

The purpose of this research was twofold. The first was to use a breeding phantom to

collect, analyze, and determine normal semen parameters for alpacas as well as to

investigate the feasibility of artificial insemination in that species. The second part of this

research was conducted using postcoital semen samples to determine the semen parameters necessary to achieve pregnancies in an alpaca. To date there has been very little research conducted that investigates the characteristics of alpaca semen.

Prior research performed by Vaughan in 2003, Morton in 2008, Wiggin in 2009, and

Mainini in 2010 has proven that collection of alpaca semen is possible with an artificial vagina placed inside a breeding phantom. The problem is that all of these studies have had varying success rates between different males; also the majority of these studies have been conducted with a small sample size. The benefit of being able to collect males using a breeding phantom and artificial vagina is that semen of breeding males can be collected and evaluated prior to breeding to determine their reproductive soundness and to identify any potential fertility problems. Additionally if these males could consistently be collected there is potential for extension of the semen and using it for artificial insemination.

Artificial insemination (AI) in other domestic livestock has been successfully used to propagate and preserve superior genetics and increase the availability of superior genetics to breeders. For the alpaca industry this could potentially improve fiber, fertility, conformation, and disease resistance in offspring. Additionally AI would eliminate the need for owners to transport males or females for matings. This would reduce the risk of spreading infectious disease, decrease stress to the animals, and increase farm biosecurity.

The problem with AI in alpacas to date is that males have proven difficult to consistently collect. This might be due to the dribbling and viscous nature of the ejaculate but the exact reason it is unknown. This study aimed to determine if a reliable and consistent technique of semen collection is even possible.

There is no research correlating semen parameters with achieved pregnancies in alpacas. If specific parameters necessary for conception could be determined then males could be evaluated for potential fertility problems before owners of males and females invested money into breedings. This would effectively increase conception rates and decrease the risk of using a stud that is subfertile.

MATERIALS AND METHODS

Animals

In this study twenty adult Huacaya male alpacas were used (Table 1). They ranged from 2.5 years to 11 years in age with a mean age of 5.4 years. Nine of the twenty males (45%) had experience with natural breeding before; the other 11 (55%) had no prior breeding experience at all. All of the males were borrowed from farms in Western Massachusetts and transported to the UMass Hadley Farm in Hadley, Massachusetts with the exception of one that was owned by the University of Massachusetts and already resided at the farm. All of the males were used for semen collection and evaluation using the breeding phantom and an artificial vagina. Additionally, males B, D, F, K, L, M, N, and P were used in natural service for correlating semen parameters from post‐breeding collections with achieved pregnancies. The males were kept at the University of Massachusetts Amherst, Hadley farm in small groups and were fed ad libitum second cut hay, fresh water, and a specially formulated mineral mix for alpacas. Season permitting, they also had access to ad libitum 2 grass pasture and pelleted grain when necessary to maintain normal body condition. They were housed in separate but adjacent pastures to the females being used for the semen parameter to pregnancy correlation study. These same females were occasionally used to stimulate the libido of the males to persuade them to mount the breeding phantom.

The study females (Table 2) were housed under the same conditions as the males. They ranged from 3 to 11 years of age and 0 to 9 previous crias. The females were used for natural service breedings and collecting postcoital semen samples, which were then evaluated with respect whether or not a pregnancy was achieved. The females were also used as teasers for the males to increase libido during semen collections using the phantom.

Physical Examination Prior to Breeding

Upon arrival at the farm all of the body condition scores of the borrowed males were assessed, and fecal samples were taken from each animal and analyzed for intestinal parasites. Every male was assessed to be in good health and adequate body condition at the start of the study. Prior to breeding all males’ reproductive organs were examined using a 7.5MHz linear ultrasound probe and their testes were measured using calipers. The ultrasound was used to visualize the size and echotexture of the left and right bulbourethral glands, the prostate gland, and both testes. Testicle length and width were measured using calipers and carefully rotating the testicle horizontally in the scrotum.

(Figure 1) Table 1 shows the general information about the males used in the trial along with the caliper measurements of their testicles. The average size for the left testicle was

4.2 cm long by 2.8 cm wide with a range of 3.8 cm to 5.0 cm long by 2.2 to 3.8 cm wide. The

3 average for the right testicle was 4.5 cm long by 2.9 cm wide with a range of 3.4 cm to 4.8 cm long by 2.4 to 4.0 cm wide. Figure 2 shows the homogenous appearance expected when viewing a normal testicular parenchyma using ultrasound. Figure 3 shows the ultrasound image of the testicular parenchyma when testicular cysts are present. All males in this study with the exception of Male T had normal homogenous appearing testicular parenchyma in both testes. Male T had hypoechoic regions in both left and right testes, which are consistent with testicular cysts.

Figure 1: Testicular measurement (length) using calipers (Pindar and Purdy)

Table 1: Study Males

Age Left Testicle Right Testicle (length x Natural Breeding Male (years) (length x width) cm width) cm Experience

A 6 ‐ ‐ yes B 4 3.9 x 2.2 3.4 x 2.7 yes C 3 4.2 x 2.6 4.1 x 2.4 no D 8 5.0 x 2.8 4.8 x 2.7 no E 6 3.9 x 2.1 3.8 x 2.9 no F 6 4.0 x 3.1 4.1 x 3.1 yes G 6 3.8 x 3.0 3.9 x 2.8 no H 5 4.1 x 2.9 3.9 x 2.7 no I 5 4.1 x 2.9 4.6 x 3.9 no J 5 4.0 x 3.1 4.6 x 3.9 no K 8 4.7 x 2.6 4.6 x 2.6 yes L 4 4.5 x 2.7 5.3 x 3.1 no M 3.5 3.8 x 2.3 3.6 x 2.2 yes N 3 4.4 x 2.4 4.5 x 2.3 no O 11 3.9 x 2.4 3.9 x 2.5 yes P 7 4.6 x 3.1 4.6 x 3.0 yes Q 2.5 4.1 x 3.3 4.8 x 3.2 no R 3 4.0 x 2.8 3.8 x 2.4 no S 6 4.8 x 3.8 4.8 x 4 yes T 5 4.2 x 3.4 4.5 x 3.2 yes Average 5.4 4.2 x 2.8 4.5 x 2.9 ‐ Range 2.5‐11 3.8‐5.0 x 2.2‐3.8 3.4‐4.8 x 2.4‐4.0 ‐

Table 2: Study Females

Age Female Number of successful pregnancies in past (years) a 2.5 0 b 3 1 c 5 2 d 7 1 e 5 1 f 13 3 g 14 7 h 11 9

Figure 2: Normal testicular ultrasound appearance

Figure 3: Abnormal testicular ultrasound appearance consistent with testicular cysts

Alpaca Collection Phantom

Previous researchers (Wiggin and Purdy, 2009) constructed the breeding phantom from a foam deer archery target, foam padding, and a tanned alpaca hide cover. (Figure 4) These were then secured to a piece of plywood using metal brackets. A section of the posterior of the archery target was hollowed out and a PVC pipe was placed inside to hold the artificial vagina (AV). (Figure 5) A few alterations were made after the original construction to allow 6 males a more natural breeding position and easier time of gripping the phantom while mounted (Mainini). In 2011 the posterior end of the phantom was slimmed down and angled in a more downward manner to compensate for the smaller sized animals being used in this phase of the trial. Also a piece of foam was taped over the opening of the PVC pipe and a hole was cut for the AV to minimize the chances of the males penetrating into any area other than directly into the artificial vagina.

Figure 4: Lateral view of phantom (Wiggin and Purdy)

Figure 5: Posterior view of Phantom (Wiggin and Purdy)

Artificial Vagina

An eight‐inch artificial vagina (AV) designed for sheep (Agtech Inc., Manhattan, KS) was cut

down to six inches length and then lined using a cylindrical latex rubber liner. Both ribbed

and smooth latex rubber liners (Agtech Inc.) were used throughout the trial but there was

no observed effect on ejaculation success or semen volume. An Agtech Inc. conical latex

rubber cone liner was then stretched over the end of the AV and secured with rubber

bands. A 15mL sterile plastic collection tube was then attached to the smaller end of the

conical liner and was also secured with a rubber band (Figure 6). To pressurize the AV the

water valve was removed and the apparatus was filled with water at approximately 45 °C

using a funnel until it began to overflow. Excess air was relieved from the liner, the water

valve was replaced, and the AV was pressurized using a rubber bulb apparatus until firm.

The AV was then kept at the required temperature of approximately 40 to 45°C one of two

ways: wrapped in an electric heating pad, or wrapped in a hot/cold pack heated for two

minutes on high in a microwave. Both methods worked adequately. The electric heating

pad was used more in the winter months because it maintained the temperature of AV

better in the colder temperatures. During the summer the heated hot/cold pack

maintained the temperature and allowed for the phantom to be more portable. The AV

wrapped in the heating apparatus was then inserted into the PVC pipe in the back end of

the phantom. Sometimes extra foam was needed to stabilize the AV under the rear end of

the phantom and to maintain the proper male breeding angle.

Figure 6: Artificial Vagina (AV) (Mainini)

Semen Collection

Phantom Training Process

The males varied tremendously in the amount of effort and time required to train them to mount the phantom and ejaculate into the AV. Some males mounted the phantom right away the first time and others were frightened by it and would not mount at all. All males were given a trial period of at least 5 mounts or attempted mounts of the phantom. If after these five plus tries they were not mounting or ejaculating into the AV they were eliminated from the trial and replaced with a different male. If the male did not commence orgling (gargling noise made by a male alpaca when sexually stimulated) and mounting the phantom immediately, a receptive female was used to attempt to stimulate the male’s libido. The male was allowed to mount the receptive female right next to the phantom until she assumed the normal sternally recumbent (kushed) breeding position. At this point the male was moved off of the live female and onto the phantom. (Figure 7) Usually after 1‐2 times of using this technique the male would learn to mount the phantom immediately without the aid of a receptive female. (Figure 8) 9

Figure 7: Male copulating with phantom next to kushed female (Mainini)

Figure 8: Male copulating with phantom unaided

Semen Collection Using Phantom

Once properly trained to mount the phantom males were collected 1‐3 times per week using the breeding phantom and artificial vagina. The phantom was placed inside the barn during inclement weather or on grass during dry days. For males needing extra stimulation the phantom was placed in the field or pen containing female alpacas to

increase libido. After assuming the copulatory position, the penis was checked for proper

positioning and was guided into the AV if assistance was required. The males were allowed

to breed for 15‐20 minutes or until they voluntarily dismounted and would not remount.

The copulation time was limited to 20 minutes to prevent a possible decrease in semen viability due to time spent exterior to a live alpaca vagina and uterus. Throughout the

period of copulation the male’s penile positioning was frequently checked and readjusted if

not correct. Some males required adjustment to the height of the phantom, which was done

by placing it onto a lower surface than the male or by raising the height of the phantom by

putting rugs under the caudal end. Once copulation ceased the males were returned to

their pen and the AV was immediately removed from the phantom and brought indoors.

When inside the semen sample was driven into the collection tube using centrifugal force

and then the collection tube was removed from the AV apparatus. At this point the collection tube was immediately placed into an incubator heated to 37 °C to prevent cold shock to the sperm until analysis was complete.

Postcoital Semen Collection

For the semen parameter correlation with pregnancies a postcoital semen sample was analyzed. The sample was obtained by inserting a plastic speculum into the vagina of the restrained, sternally recumbent female immediately post copulation. Gentle scooping of the speculum along the ventrum of the vagina near the cervical opening permitted a semen sample to be easily obtained from the vagina. The sample was then stored in an incubator at 37 °C until analysis was complete.

Semen Analysis

Both the semen collected from the phantom breedings and the postcoital samples were analyzed for the same parameters using the same methodology. Estimated sperm activity percentage, estimated sperm concentration, sperm morphology, and percentage of live sperm were all evaluated. The only parameter assessed for the phantom breedings that was not available for the postcoital samples was volume (in milliters) of the ejaculate, since the sample obtained was only that which overflowed or was carried into the vagina as the male dismounted.

Volume of Ejaculate

For the samples collected using the phantom the volume of ejaculate was measured immediately following centrifugal forcing of the ejaculate into the collection tube. It was measured using the graduated scale on the side of the collection tube. Only the liquid portion of the sample was measured; any foam was not included. Sometimes the sample had to be left in the incubator for a few minutes so the foam could settle in order to get an accurate reading.

Estimated Sperm Activity

Sperm activity was used as a quantitative measure of the viability of the sperm. It was determined by placing a drop of fresh semen onto a pre‐warmed microscope slide with pre‐warmed cover slip applied on top. This slide was then observed at 400X total magnification and the sperm were viewed in ten different locations on the slide. From these ten different locations an estimate of how many sperm were moving versus how

12 many were not was made for every ten sperm observed, and an estimated average percentage of active sperm was thus determined. The viscous nature of alpaca semen decreases the amount of progressive motility dramatically and thus the amount of oscillatory activity (movement in place) was instead assessed.

Estimated Sperm Concentration

Concentration of the ejaculate was estimated using a similar technique to the sperm activity estimate. Ten fields on the same slide used to determine the sperm activity were viewed and a value of 1, 2, or 3 (low, medium, or high) was subjectively assigned, which represented the average concentration of the sperm on the slide. (Figure 9) It was important to view ten different fields on the slide because concentration and activity of sperm varied depending on the location. If more than 6 out of the 10 areas viewed, were of high concentration then the sample was considered to be a 3 out of 3. If 6 out of the 10 areas were low concentration then the sample was considered a 1 out of 3. Anything in between was considered to be medium concentration and was assigned a value of 2 out of

Figure 9: Low, Medium, and High Concentrations (from left to right)

Sperm Percent Live and Morphology

A drop of fresh, immediate post collection semen was placed on a warmed slide along with

a warmed drop of eosin‐nigrosin stain. Another warmed slide was then placed on top of

the stain and semen mix and then dragged apart in order to create a smear. The two slides

were then left to dry before observation. Using 1000X magnification and a drop of immersion oil, 200 sperm were counted. From these 200 sperm the number of live sperm seen was determined and then a percentage live was recorded. Sperm cells that are alive at the time of staining do not absorb the die as well as dead sperm and thus they appear much lighter in color than dead sperm. (Figure 10)

The sperm morphology was determined from the same 200 sperm sample on the slide prepared as described above. Proximal and distal cytoplasmic droplets, bent or severely coiled tails, midpiece defects, decapitated heads, separated heads and tails, and abnormal heads were all morphological defects that were characterized as abnormal. These defects all are considered as severe enough to prohibit proper function of the sperm and therefore to decrease or completely prevent fertilization of an oocyte.

Figure 10: Live/dead stained alpaca sperm (Mainini)

RESULTS AND DISCUSSION

Effect of Photoperiod Length on Sperm Activity and Live Sperm Percentage

In some species photoperiod has a large effect on reproduction, including male libido,

testicle size and sperm morphology. (Tibary 2005) However, previous work at the

University of Massachusetts indicated that photoperiod had no effect on sperm

morphology in alpacas. (Mainini, 2010) One of the objectives of this study was to investigate if there would be a difference in sperm activity or live sperm percentage

depending on the time of year collected. Data was collected over a period of 11 months in

2011. (Table 3) Fourteen different males were collected in Massachusetts using the

breeding phantom. From each collection the sperm activity and percent live were evaluated

and calculated. It is important to note that the same 14 males were not collected every

month so there is some margin of error because of individual male variation. However,

when males are analyzed individually the same amount of inconsistency in percentages is

seen suggesting that the variation is not due to photoperiod but instead is normal for

alpacas. Estimated sperm activity percentages ranged from 0 to 96%. Maximum and

minimum values were randomly distributed and not associated with a particular calendar

month or length of daylight. The same was found for live sperm percentages with a range

of 0 to 97.5%. Upon compilation of the data there was no indication that photoperiod had

any effect on sperm activity or percent live. (Figures 11 and 12)

Figure 13 shows this inconsistency on an individual level. Male S was collected 11 times

over a three‐month period using the breeding phantom and AV. Granted the hours of

daylight only ranged from 11.2 hours on October 11th to 9.1 hours on December 29th but

15 still the variation from collection to collection was significant and appeared random.

Sperm activity percentages ranged from 0 to 95% with neither the peaks nor zero activity collections corresponding to a particular time of year.

Figure 11: Sperm Activity vs. Calendar Month of Collection

Sperm Activity vs. Calendar Month of Collection 100 80 Activity(%) 60 40 Sperm 20 0 024681012

Estimated Calendar Month of Collection (n=14 animals)

Table3: Average Monthly Ranges in Hours of Daylight

Range of Month # Calendar Month Photoperiod Length (hours) 1 January 9.1‐9.9 2 February 10‐11.1 3 March 11.1‐12.5 4 April 12.5‐13.8 5 May 13.8‐14.7 6 June 14.8‐15 7 July 14.2‐14.9 8 August 13‐14.2 9 September 11.6‐13 10 October 10.3‐11.6 11 November 9.3‐10.2 12 December 9‐9.3

Figure 12: Live Sperm vs. Calendar Month of Collection

Live Sperm vs. Calendar Month of Collection 100

60 Sperm(%)

Live 20

0 024681012 Calendar Month of Collection (n=14 males)

Figure 13: Sperm Activity vs. Collection Date: Male S

Sperm Activity vs. Collection Date: Male S

100

60 Activity(%)

50 Sperm 40

Estimated 20

0 5‐Oct 25‐Oct 14‐Nov 4‐Dec 24‐Dec 13‐Jan Collection Date (2011)

Consistency of Phantom Collections:

The main limitation for advancement of the use of artificial insemination in alpacas has

been the inability to successfully collect the ejaculate consistently. (Wiggin 2009, Mainini

2010) One of the purposes of this study was to see if the inconsistency with collections among males was due to the small sample sizes in prior trials or if this inconsistency would hold true in a larger trial. The results of this trial were consistent with those of prior trials.

The overall success rate (which was defined as number of collections with a volume of

ejaculate over 0.1 mL and more than 5 sperm seen on the slides used to determine activity

or morphology) was only 38%. (Table 4) When viewed in the context of males with prior

breeding experience versus males with no prior experience the numbers were relatively

consistent. For males with prior breeding experience the success rate was slightly higher

at 40% than the 35% success rate of males with no prior experience. However, when

activity percentages of the successful collections as defined above are factored in the

numbers are essentially equivalent. The success rate with active sperm for males with

previous breeding experience is 34% and for males with no prior experience is 31%. The

overall success rate with active sperm was 33% for all mounts. Table 4 shows these rates

and also shows the breakdown for each individual animal.

Of 101 total mounts on the phantom there were only 38 successful collections, a 38%

overall success rate. Out of the 45 collections in which activity percentages were recorded,

only 15 were successful with an activity percent greater than 0. Therefore the percentage

of productive collections was only 33%. Based on these numbers and the variability seen

above (Figures11, 12, and 13) it can be concluded that with this current technique artificial

18 insemination does not seem to be a viable assisted reproductive technology for alpacas.

The data from this trial and previous trials suggests that not every male can be successfully collected during every attempt using the breeding phantom and artificial vagina technique used in this trial. The reason for the variations observed in semen parameters among males collected at different times of the year could not be determined. Therefore, economically it would not be profitable for producers to pay to have their male trained and collected with no guarantee of a marketable product (semen) each time it would be needed to inseminate a female.

Table 4: Semen Collection with Phantom Success Rates

Number of Success Rate Number of Times Success Successful* with Age Number of Successful* Male Mounted Rate collections Active (yrs) Collections on Phantom (%) with Sperm Activity%>0*** (%) Males With Previous Breeding Experience: M 3.5 10 4 40 3 30 B 4 7 0 0 ‐ _ T 5 8 7 88 2 25 F 6 2 0 0 ‐ _ S 6 11 6 54 5 45 A 6 2 1 50 ‐ _ O 11 2 0 0 ‐ _ P 7 4 0 0 ‐ _ K 8 7 3 43‐ _ Section Avg. 53 21 40 10 34 Totals 6.3 Males Without Previous Breeding Experience: Q 2.5 6 3 50 1 17 C 3 8 4 50 ‐ _ N** 3 1 0 0 ‐ _ R 3 6 2 33 2 33 L** 4 1 0 0 ‐ _ I 5 4 0 0 ‐ _ H 5 4 2 50 2 50 J** 5 1 0 0 ‐ _ G 6 4 0 0 ‐ _ E 6 9 6 67 ‐ _ D 8 4 0 0 ‐ Section Avg. 48 17 35 5 31 Totals 4.6 Overall 101 38 38 15 33 Totals *Successful= volume>0.1 mL and >5 sperm seen on activity and morphology slide ** Did not mount dummy successfully several times so was traded for a different male *** Only animals where activity percentages were recorded for each collection are reported 19

Volume of Ejaculate

Another problem with the use of artificial insemination in alpacas is the inconsistency in

ejaculate volume collected with the phantom. For example, Male T ejaculated 2.0 mL of

semen into the AV the first time he was collected, then 3.5 mL the second, then only 0.5 mL

the third collection, 0 mL the fourth, but then 0.75 mL again for the sixth collection. These

discrepancies in semen volume collected are seemingly sporadic and do not seem to be

affected by age of animal or photoperiod, but they are a consistent trend throughout the males used in this trial. (Figure 14) The frequency of male use could affect the volume of the ejaculate but most of these males were not used more often than once every 2‐3 days.

This inconsistency once again makes investing in artificial insemination unprofitable for the owners of the males. If they were to pay to have their male trained to mount the phantom there would be no guarantee how long it would take to achieve any volume of ejaculate. It is not known how many normal sperm are necessary to successfully artificially inseminate a female but if the chances are high that it will not be profitable to the owners of the male it would not matter.

Figure 14: Semen Volume Collected vs. Collection Number

Semen Volume Collected vs Collection Number 4

3.5

2.5 (mL) Male C 2 Male E Volume Male M 1.5 Male S Semen Male T 1

0.5

0 024681012 Collection Number

Sperm Morphology

Prior studies have resulted in different conclusions about what morphological defects are

most prevalent in alpacas and what the overall percentage of morphological abnormalities

is for a typical male alpaca. Vaughan and Tibary’s research concluded that head

abnormalities were the most common defect (Tibary 2006) but a trial performed at the

University of Massachusetts and also by Bravo found that tail defects were the most

common. (Bravo 1997 and Mainini 2010) One of the goals of this study was to increase the number of study animals and number of collections using the phantom to try to determine

21 a more accurate average and range for sperm morphology. Morphological abnormalities and defects are important because a deformed or immature sperm cell will have a decreased or no chance of fertilizing an oocyte. Sperm cells in this study were characterized as normal, (Figure 15) or determined to have a significant morphological defect or abnormality. Data was collected and analyzed from 47 phantom collections.

Cytoplasmic Droplets

Cytoplasmic droplet (Figure 16) presence, either proximal or distal to the head along the midpiece, is an indicator of sperm cell immaturity. These sperm cells were found in every alpaca ejaculate studied. During the natural maturation process the proximal and distal droplets would be shed if they remained within the male reproductive tract longer. They were not counted as normal in this trial because they are not considered to have the capability to fertilize an oocyte, which has been shown by a decreased binding to the zona pellucida and an inability to capacitate in a study conducted with canines (Pena 2007).

Additionally in bulls proximal droplet incidence decreased after the onset of puberty (Pena

2007) and in the earlier study conducted at the University of Massachusetts the males who were 4, 5, and 6 years of age had the highest incidence of cytoplasmic droplets (n=8; age range 4‐15 years; mean 6.8 years) (Mainini 2010). This was hypothesized to be due to the fact that these males were still undergoing puberty.

In this study, males E, P, R, S, and T had the highest average percentages of cytoplasmic droplets when both proximal and distal droplets were combined. These males were 6, 7, 3,

6, and 5 years of age respectively. Male S at 6 years of age averaged 13.1% proximal droplets and 6.1% distal droplets per collection, based on nine collections. Male M at only

3.5 years of age averaged only 4.6% proximal droplets and 0.9% distal droplets per collection, based on 7 collections. (Table 5) This suggests that cytoplasmic droplet percentage is not directly related to puberty. Based on the 47 collections and 10 males with morphological data in this study it appears that cytoplasmic droplets are normal in alpacas. Unpublished data from the southern Peruvian highlands (Purdy et al, 2010‐2012) also showed these sperm to be common in every male sampled post natural service breeding to date (n=16).

Tail Abnormalities

There were three different classifications of tail abnormalities: bent, severely coiled, or tailless. (Figure 17) Bent tails were classified as having a bend in the flagella that was greater then or equal to 90 degrees; anything less was considered to be normal positioning at the time of staining. Severely coiled tails referred to the sperm where the tail began curling immediately adjacent to the midpiece. Sperm where coiling was not as tight and commenced at the caudal end of the tail was most likely due to cold shock and were counted as morphologically normal. Tailless sperm were just a head with no flagella attached. All of these tail abnormalities would cause the sperm to be unable to move properly swim and thus render it incapable of fertilizing an oocyte. From the data collected in this study on average 11.1% of the sperm in each ejaculate had some sort of tail abnormality. This defect was the most common in the study animals.

Head Abnormalities

Abnormal head shape, headless sperm, and decapitated heads were the three classifications of head abnormalities observed in this trial. (Figure 18) Abnormal head 23 shapes included microcephalies (small heads), piriform heads, blunt heads, and double‐ headed sperm. Headless sperm were those in which the head was completely severed and disconnected from the midpiece and the tail. Decapitated heads referred to sperm in which the head was still attached to the midpiece and tail but the head was tilted back towards the midpiece. Any abnormality to the head of a sperm cell will prevent proper fertilization.

For this study there was an average of 4.0% sperm per ejaculate with one of the three head abnormalities listed above. This number is significantly lower than the amount of tail abnormalities, and is consistent with the results reported by Mainini in 2010 but is inconsistent with Vaughan and Tibary’s study.

Midpiece Defects

A rough, broken, thickened, or bent midpiece were all classified under midpiece defects.

(Figure 19) A bent midpiece was a bend that was significant enough that it was not just the result of the sperm being stained in flagellar motion. Any type of defect to the midpiece most likely inhibits the proper motility of the sperm and decreases its chances of being able to properly fertilize an oocyte. On average 3.7% of sperm per ejaculate showed a midpiece defect in this study.

Figure 15: Normal Sperm (Mainini 2010)

Figure 16: a) Proximal droplet; b) Distal Droplet (Mainini 2010)

Figure 17: Tail Abnormalities: a) Tailless; b) Severely coiled; c) Bent (Mainini 2010)

Figure 18: Head Abnormalities: a) Decapitated head; b) Double‐headed; c) Headless;

d) Abnormal head (misshapen) (Mainini 2010)

c d

Figure 19: Midpiece Defect: a) Broken midpiece b) Bent midpiece

Table 5 shows the compilation of all the data for the 47 collections. It is organized by

animal; the number of collections per animal is also indicated. The total average

morphologically normal sperm per ejaculate was 72.6% with a wide range of 31.0‐92.5%.

This indicates that morphology of sperm ranges widely from collection to collection. This

data is consistent with Mainini’s study in 2010 in which she observed the range of

morphologically normal sperm to be 35.1‐88.4% (Mainini 2010) and differs from the Bravo

study conducted in 1997 in which he determined the morphologically normal range to be

the same as it is in other livestock species (70.6‐84.1%.) Furthermore, it can be easily seen

that not only does morphology vary widely among different males, but also among

individual collections in males themselves for each male studied. For instance, over the

course of 7 collections using the breeding phantom, male E had an average morphologically normal sperm percentage of 68.9% but a range of 31.0‐87.5%. The same is also true for male S who over a course of 9 collections averaged 62.8% morphologically normal sperm with a range of 51.0‐91.5%. This variation is consistent with the variation seen among

sperm activity percentages and live sperm percentages. (Figures 11, 12, and 13)

Table 5: Sperm Morphology Percentages for Study Animals (Averages and Ranges)

Male # of Collections Severely Age With Morphogically Proximal Distal Bent Midpiece Decapitated Abnormal Coiled Tailless Headless (yr) Morphology Normal Droplet Droplet Tail Defect Head Heads Tail Data 59.7 3.6 4.4 5.6 2.1 2.9 13 7.4 C (3) 4 0 0 48.5‐ 70.5 1‐8.5 0‐10.5 2.5‐8.9 1.5‐ 3.5 0.5‐ 4.9 3.5‐ 25.3 1‐ 17.4

68.9 10.8 2.5 1.6 0.9 2.6 2.4 6.9 4.5 1.1 E (6) 7 31‐ 87.5 1‐18 1‐6.3 0‐ 4 0‐5 1‐ 6.3 0‐13 0‐38 0.5‐14 0‐5

86.6 4.53 2.3 0.2 3.41 2.94 H (5) 2 0 0 0 0 85‐ 88.2 3.18‐5.9 0‐4.5 0‐0.2 0‐6.8 0‐5.9

80.5 3.2 2.5 2.7 2.32 4.1 0.1 4 0.5 K (8) 4 0 74.7‐ 86.3 0‐7.5 0‐4 0.5‐5.3 0.7‐ 3.5 1.5‐8 0‐0.5 0‐13.3 0‐2

81.4 4.6 0.9 4.3 1.6 3.2 1.9 1.3 0.4 0.7 M (3.5) 7 60.5‐ 95.2 0.9‐ 9 0‐3 0.1‐9 0‐4 0.5‐7.6 0‐9 0‐3.3 0‐1 0‐2

84.8 7.5 3.2 1.9 2.4 0.4 P (7) 2 0 0 0 0 78.7‐91 3‐12 0‐6.5 0.9‐3 1.9‐3 0‐0.9

75.5 12 1.3 2.3 5.3 5.5 0.8 0.5 1.2 1.5 Q, (2.5) 3 69.5‐85.5 3‐19 0.5‐2 1‐3.5 5‐6 4.5‐7 0‐2.5 0‐1 1‐1.5 1.5‐1.5

76.6 11.8 3.9 6.2 0.9 5 R (3) 2 0 0 0 0 66‐87.3 10.5‐13 1.8‐6 4‐8.3 0.9‐1 0‐10

62.8 13.1 6.1 2.6 8.2 4.6 0.5 1.2 0.5 0.5 S (6) 9 51‐91.5 0‐25.1 0‐20 0.5‐7 0‐17 1‐7.5 0‐1.6 0‐5 0‐3 0‐3.5

49.2 10 1.1 4.9 14.1 10.4 4.3 3.1 2.9 1.6 T (5) 7 40‐57 3.5‐20 0‐3 0‐14.5 5.5‐24.5 7.5‐13 0‐16 0‐8 0‐15 0‐3.3

72.6 8.1 1.9 3.1 4.5 3.6 1.0 3.5 2.1 0.8 Average n= 47 31‐ 95.2 0‐25.1 0‐20 0‐14.5 0‐24.5 0‐13 0‐16 0‐25.3 0‐17.4 0‐5.9

27 Semen Parameter Correlation with Achieved Pregnancies

The purpose of the other part of this study was to correlate semen parameters with pregnancies and determine what semen characteristics quantitatively are sufficient to achieve a pregnancy in alpacas. Based on the variability of numbers observed in phantom collections it was hypothesized that the same variability and wide range of values would hold true for postcoital semen samples. It is unknown which parameters are most important for achieving pregnancies it was completely unknown but this study investigated estimated sperm activity, estimated sperm concentration, live sperm percentage, and morphologically normal sperm percentage and compared these values with whether or not a pregnancy was achieved. Pregnancy achievement was determined using transrectal ultrasound examination by visualization of an embryonic vesicle in the female at any point post copulation. Embryonic vesicles were usually seen between 9 and

20 days after breeding.

The results of this trial were fairly inconclusive. The values for breedings in which pregnancies were established were slightly higher than those for the breedings that did not result in pregnancies with the exception of concentration in which the averages were similar. (Table 6) The average estimated sperm activity percentage for breedings resulting in pregnancies was 59.3% compared to the 56.7% average for breedings in which pregnancies did not occur. For percent live sperm there was a slightly greater difference; for breedings resulting in pregnancies the average was 87.0% whereas for those breedings not resulting in pregnancies it was only 82.9%. However, when the ranges are compared for both achieved and non‐achieved pregnancies, they are comparable to each other, thus

28 suggesting once again that semen parameters in alpacas vary widely from male to male and collection to collection. This also suggests that even with such variation in semen parameters pregnancy can still be achieved. It makes sense that the sperm activity percentages and live sperm percentages would be greater in breedings that resulted in pregnancies because the more live and active sperm in an ejaculate the higher the chance of one sperm being able to reach, penetrate, and fertilize an oocyte. However, the differences seen from the breedings in this trial were not significant enough to determine what exact values for percent active sperm and live sperm are necessary to achieve a pregnancy. For the breeding of male O to female h, the estimated activity percentage was observed to be 0%. The reason for this is most likely that the sample was viewed after all of the sperm had become inactive.

When the percentage of morphologically normal sperm was analyzed for this portion of the study the results were similar. For breedings that achieved pregnancies the percent of morphologically normal sperm was 76.6% compared to 74.5% in breedings in which pregnancies were not achieved. When the range of values was examined a large difference between the two pregnancy results categories was found. In breedings in which pregnancies were established the range of morphologically normal sperm was only 60.5 ‐

88.2%. The range for morphologically normal sperm in breedings in which pregnancies were not established was much larger, 0 ‐ 92.5%. These values could indicate that on the lower end of the range ejaculates with less than 60.5% of morphologically normal sperm cannot achieve pregnancies in alpacas or it could be due to the fact there were more collections from breedings in which pregnancies were not established. More research is needed to validate this conclusion. 29

The females used in this study also contribute to the lack of conclusiveness in the data.

Females f and g are 13 and 14 years old respectively and most likely did not conceive not because of faulty semen but because they are old and might not have any viable ovum left.

Similarly Female a was only 2.5 years at the time of this trial and may not have reached complete sexual maturity and thus did not conceive. The females were also being ultrasounded and behavior tested every to every other day so may not have conceived due to stress. To more accurately describe what semen parameters are necessary for conception a larger trial with more females and more breedings, with the same males, would need to be conducted.

Table 6: Semen Parameters from Live Breedings and Pregnancy Results

Estimated Concentration % Estimated % Live Pregnancy Male Female 1=low Morphologically Activity % Sperm Achieved 2=med Normal 3=high B a 10 3 88.5 72 no B d 80 3 82.5 60.5 yes D c ‐ 1 87.9 57.6 no F b 50 3 99.5 78 yes F e ‐ ‐ 66 74 no F f 30 1 ‐ 0 no F c 70 3 75 75 no F b 80 1 69.1 88.2 yes F f 70 3 80 83 no F d 50 2 65.2 84.8 no M e 80 3 ‐ ‐ yes M g 60 2 94 87 no L g 85 3 ‐ ‐ no N g 50 2 81.5 86 no K c ‐ 3 94.2 82.2 yes K e 70 3 89.9 91.6 no K a 75 3 92 92.5 no K b 65 ‐ 93 85 yes P g 80 3 91.5 90.5 no P d 60 2 86 77 yes P e 30 2 ‐ ‐ no P a ‐ 1 ‐ ‐ no O h 0 2 79 80 yes Q h ‐ ‐ 93 62 yes Avg/ Range 59.3 2.4 87 76.6 9 for (0‐80) (1‐3) (69.1‐99.5) (60.5‐ 88.2) pregnancies pregnancies

Avg/ Range 15 56.7 2.3 82.9 74.5 for non‐ non‐ (10‐85) (1‐3) (66‐94) (0‐92.5) pregnancies pregnancies

Total Avg/ 57.6 2.33 84.6 75.3 24 total

Range (0‐85) (1‐3) (66‐99.5) (0‐92.5) collections

FUTURE WORK

As for the potential of artificial insemination in alpacas this technique is not profitable for all owners of the males. There are too many variables that that affect successful collections of individual males to make it worthwhile to owners, or to make it worthwhile to continue this study. There is opportunity to conduct additional research using the phantom with males that can be trained to mount the phantom and ejaculate into the artificial vagina consistently. With these males more research could be done to determine whether or not photoperiod does have an effect on any of the various characteristics of the semen. A trial should be conducted using a larger group of males for an entire 12 months to determine if there is any variation in any of the semen parameters, specifically volume collected, percent live and active sperm, concentration of sperm, and morphological defects. If it were to be determined that there is a decrease in male fertility during any of the seasons this could help owners maximize their production by not even attempting to breed their females during this period. Using males that consistently mount and ejaculate into the phantom, a study could also be performed to determine the effect of consecutive day breedings on fertility. A study of this nature could be used to ascertain the proper management for breeding males. Currently it is thought that males should not be bred multiple days in row because of a decrease in fertility in the later days. Data to determine how many days in a row or how many breedings a day a male can perform without a decrease in fertility could help owners to better manage their breeding males and increase conception rates among females. However it is suspected that as was true with other semen parameters, this will fluctuate among males. This fact even now demonstrates the need for stud owners to frequently assess the semen production of their males. Lastly,

32 further research with an increased sample size also needs to be completed to conclusively determine what ranges of semen parameters are necessary to impregnate an alpaca. This trial has provided some initial data but a larger one using a more consistent group of females is needed to accurately determine these parameters. A trial using, females that are known to be fertile and the same males for multiple breedings would be necessary to decide upon the exact range of parameters needed to achieve a pregnancy.

AKNOWLEDGEMENTS:

I would like to thank Dr. Stephen Purdy and Dr. Carlos Gradil for their assistance and expert advice during this study. I would also like to thank Caitlin Donovan, Lauren Melle,

Meghan Gennings, Megan Bernard, Melissa Wilk, Danielle Youngman, Robyn McNeil, and

Weston Brown for their assistance in collecting and analyzing the samples. Additionally I would like to thank Craigieburn Farm Alpacas, Great Rock Alpacas, and Maple Brook Farm

Alpacas for the use of their males.

References

Bravo, W., et al. “Collection of Semen and Artificial Insemination of Alpacas” Theriogenology. 47 (1997) 619‐626.

“Abnormalities of Reproduction in Lamoids” The Reproductive Process of South American Camelids. Salt Lake City: Seagull Printing, 2002. 97‐99.

“Male Reproduction.” The Reproductive Process of South American Camelids. Salt Lake City: Seagull Printing, 2002. 49‐ 58.

Morton, K., et al. Continued Development in Artificial Insemination Technology in Alpacas. RDIC 2008.

Mainini, T., S. Purdy. Semen Evaluation and Extension in Alpacas (Vicugna pacos). University of Massachusetts, Amherst, 2010.

Pena, AI., et al. “Infertility in a Dog due to Proximal Cytoplasmic Droplets in the Ejaculate: Investigation of the Significance or Sperm Functionality In Vitro” Reproduction Domestic Animals. 42 (2007) 471‐478.

Sumar, J. “Reproduction in llamas and alpacas” Animal Reproduction Science. 42 (1996) 405‐415.

Tibary, A., J. Vaughan. “Reproductive physiology and infertility in male South American camelids: A review and clinical observations” Small Ruminant Research. 61 (2005) 283–298.

Vaughan, J, et al. Artificial Insemination in alpacas (Lama pacos). RIRDC, 2003.

Wiggin, M., S. Purdy. Semen Collection and Evaluation in Alpacas (Lama pacos). University of Massachusetts, Amherst, 2008.