Essential guides. Semen collection and processing

PRESENTATION

BROCHURE ESSENTIAL GUIDES ON SWIN

HEALTH AND PRODUCTION

S men collection and processing Carlos García Artiga Coordinators: Rosa María García García María Arias Álvarez

ESSENTIAL GUIDES ON SWINE HEALTH AND PRODUCTION

Semen collection and processing

ESSENTIAL GUIDES ON SWIN

HEALTH AND PRODUCTION

S men collection and processing Carlos García Artiga Coordinators: Rosa María García García María Arias Álvarez

AUTHOR: Carlos García Artiga.

COORDINATORS: Rosa María García García and

María Arias Álvarez.

FORMAT: 17×11 cm.

NUMBER OF PAGES: 82.

NUMBER OF IMAGES: 67.

BINDING: Paperback, wire-o.

RETAIL PRICE

35 €

Artificial insemination (AI) is an indispensable technique on swine farms, and its success depends primarily on semen quality. This handbook covers the key steps necessary to obtain high-quality processed semen and ensure successful AI. Essential practical considerations are clearly detailed in each chapter.

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Semen collection and processing

Presentation of the book The collection, evaluation, dilution, and processing of boar semen are critical stages in swine AI. Artificial insemination involves the recovery and use of ejaculate from one or several boars, and subsequent processing to increase the ejaculate volume, thus allowing the insemination of multiple sows. This requires the recovery of ejaculate from individual trained boars, and processing of the semen to obtain high-quality doses. Applying this strategy ensures optimal use of the boar and improves fertility rates on the farm.

To ensure optimal reproductive performance, semen analysis (determination of the volume of the ejaculate, motility examination, estimation of concentration, and the assessment of morphoanomalies and membrane integrity) should also be performed.

This handbook offers a wealth of practical information on the main stages of AI for greater success when collecting and extending boar semen, and describes the necessary steps to ensure that gross semen evaluation on farms meets minimal quality standards.

The author Carlos GarcĂ­a Artiga He acquired his PhD in Veterinary Medicine from the University of Murcia in 1992, and in the same year became a Professor in the Zoology teaching unit of the Department of Physiology (Animal Physiology), Faculty of Veterinary Medicine, Complutense University of Madrid (UCM).

His main research focus is mammalian reproductive biotechnology, including reproductive biology of sheep, reproductive biology of pigs, spermatic characteristics in different species, freezing of pig embryos and study of porcine respiratory and reproductive syndrome.

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In addition, he is an active collaborator with several institutions, including the National Institute for Agricultural Research (CIT/INIA, Madrid), the Regional Centre for Animal Selection and Reproduction (CERSYRA), and the Department of Animal Pathology (Head of Infectious Diseases) at the Faculty of Veterinary Medicine, UCM.

Semen collection and processing

Coordinators Rosa María García García She received her PhD in Veterinary Medicine from the UCM and her Masters in Animal Production from the Mediterranean Agronomic Institute of Zaragoza (IAMZ-CIHEAM).

Currently she is a Assistant Professor in the Department of Physiology (Animal Physiology) of the Faculty of Veterinary Medicine, UCM, and a specialist in Reproductive Physiology and Biotechnologies of Reproduction. She is a lecturer on the Veterinary Medicine degree course of the UCM and on doctoral and postgraduate courses at the UCM and at other national universities (Polytechnic University of Madrid and Catholic University of Avila).

She has directed several Masters and doctoral theses in the field of animal reproduction.

She has spent time at various prestigious Spanish and European universities as a visiting researcher, and regularly participates as a researcher in competitive research projects. She is a regular reviewer for scientific journals and she has authored or co-authored numerous scientific publications in peer-reviewed journals, as well as popular science articles and book chapters.

María Arias Álvarez She received her PhD in Veterinary Medicine from the UCM (European PhD cum laude and Best Doctorate Student Award). She is currently a Professor in the Department of Animal Production at the Faculty of Veterinary Medicine of the UCM. She is a lecturer on the degree and Masters courses at the UCM, the University Alfonso X El Sabio (UAX), the Polytechnic University of Madrid (UPM), the University of Salamanca, and the University of Antwerp (UA), specialising in Animal Production, Physiology, and Biotechnologies of Reproduction.

An expert in reproductive physiology, she specialises in the study of interactions between the mother and the oocyte, embryo, and foetus in different animal species. She has collaborated with multiple institutions including the INIA, UA, and UPM, and has spent time as a visiting researcher at institutions including the UA, UC Davis (California), and University College of Dublin (Ireland). She has participated in multiple competitive research projects and has authored or co-authored numerous articles on animal reproduction published in JCR-indexed scientific journals.

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ESSENTIAL GUIDES ON SWIN

HEALTH AND PRODUCTION

S men collection and processing Carlos García Artiga Coordinators: Rosa María García García María Arias Álvarez

Table of contents 1. Introduction 2. Anatomy and physiology Anatomy Hypothalamic-pituitary-gonadal axis Spermatogenesis Reproductive development

3. Collection and training room Introduction Collection area Boar training Semen collection process

4. Method for collecting boar semen Double glove technique Sperm fractions

5. Evaluation of semen quality Introduction Volume Motility Morphology (abnormalities) Sperm concentration and sperm count Acrosome morphology Agglutination Data sheet for monitoring boars

6. Semen processing and storage Semen extenders Extender preparation Preparation of semen doses ando semen storage

7. References

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Anatomy and physiology

Semen collection and processing

Anatomy The reproductive tract of the male pig consists of several structures: the testes, epididymis, accessory sex glands, duct system, and penis (Fig. 1).

The main functions of the testes are to produce spermatozoa and secrete hormones, while the epididymis is responsible for the transport, maturation and storage of semen.

The prostate, seminal vesicles, and bulbourethral glands (Cowper’s glands) comprise the accessory sex glands, which are involved in the production of seminal plasma. Therefore, the ejaculate is composed mainly of sperm and seminal plasma.

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ANATOMY AND PHYSIOLOGY

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Figure 1. Reproductive tract of the male pig.

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1. Retractor penis muscle 2. Vesicular glands 3. Prostate gland 4. Bulbourethral glands 5. Tail of epididymis 6. Vas deferens 7. Body of epididymis 8. Testis 9. Scrotum 10. Head of epididymis 11. Spermatic cord 12. Sigmoid flexure of penis 13. Glans penis 14. Preputial diverticulum 15. Penile urethra 16. Bladder

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Anatomy and physiology

Semen collection and processing

Spermatogenesis Spermatogenesis is an extremely complex process, which involves the constant production of spermatozoa in the seminiferous tubules of the testes. In the adult boar, the testicular parenchyma consists of two compartments: tubular and intertubular compartments (Figs. 3 and 4).

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Figure 3. Histological section of the testes of an adult boar.

Tubular compartment (seminiferous tubules) containing Sertoli cells and germ cells (1), and intertubular compartment (interstitial tissue) (2) containing Leydig cells and blood vessels (10×).

Figure 4. Detail of a seminiferous tubule containing Sertoli cells and germ cells in differents stages, elongated spermatids (green arrow) and intertubular compartment containing Leydig cells (black arrow) and blood vessels (red arrow) (40×).

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ANATOMY AND PHYSIOLOGY

◗ The tubular compartment, composed of seminiferous epithelium and containing Sertoli cells and germ cells in different stages of development. ◗ The intertubular compartment, which contains a large number of Leydig cells and blood vessels.

In boars, the process of spermatogenesis takes about 35 to 40 days and transit of spermatozoa through the epididymis approximately 9 to 14 days (6–8 days in the head and body of the epididymis and storage in the tail for approximately 8 days; Fig. 5).

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Vas (ductus) deferens Caput Epididymis

Corpus

Cauda

Testis

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Figure 5. Testes and epididymis of an adult boar. The spermatozoa travel from the head (caput) to the tail (cauda) of the epididymis in a process that takes between 9 and 14 days. About 75 % of the spermatozoa contained in the epididymis is found in the tail.

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4

Method for collecting boar semen

Semen collection and processing

To collect semen, technicians should adhere to the following steps: 1. The hand that manipulates the foreskin should be covered with two vinyl gloves (one over the other).

2. The boar’s foreskin should be emptied to remove its contents, as well as any remaining urine that could contaminate the ejaculate. The preputial orifice is massaged with gauze soaked in antiseptic and disinfectant (highly diluted to avoid damaging the mucous membranes). This massaging of the foreskin stimulates pelvic movement and the expulsion of preputial fluid. The boar will be stimulated and externalise the penis.

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3. At this precise moment the first glove, which has been in contact with the foreskin, is removed.

4. Next, the hand should be closed, allowing the penis to pass through 4 or 5 times. The placement of the hand and the strong pressure exerted on the boar’s spiral-shaped penis mimics the morphology of the uterus of a sow in heat (Figs. 1 and 2). When the penis is held firmly, the boar begins to ejaculate (Figs. 3 and 4).

5. The penis must be held as horizontal as possible to prevent contamination of the ejaculate with traces of urine.

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METHOD FOR COLLECTING BOAR SEMEN

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Figure 1. Close hand around the penis, allowing the penis to pass through the hand several times when pelvic movement begins.

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Figure 2. The firm pressure exerted upon the penis stimulates ejaculation.

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5

Evaluation of semen quality

Semen collection and processing

Volume The volume of the ejaculate can be determined using a warm (35–37 °C) graduated cylinder, or by weighing on a precision scales, assuming that one gram corresponds to a volume of one cubic centimetre or millilitre (Fig. 1). The volume of the sperm-rich fraction is between 80 ml and 150 ml.

A larger volume of ejaculate does not imply a higher number of sperm.

This parameter is evaluated to determine the number of seminal doses.

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EVALUATION OF SEMEN QUALITY

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b

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Figure 1. Determination of ejaculate volume using a graduated cylinder (a) or a precision balance (b).

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Evaluation of semen quality

Semen collection and processing

Acrosome morphology The acrosome is located at the apical end of the sperm head, and contains a series of enzymes necessary for the fertilisation process. Evaluating acrosome integrity is a routine technique in specialised laboratories, and requires a phase contrast microscope. The acrosome is categorised following the classification system of Pursel and Johnson (1975), as shown in Figure 14 and described below: ◗ Normal apical ridge (NAR): the acrosome is fully adhered to the nucleus of the sperm, with a clear boundary between the two and with a distinct crescent shape at the apical ridge of the head (Figs. 14a and 15). ◗ Normal apical ridge with particles (NAR‘): as described for NAR, except for the presence of small adherent particles in the apical region and in the postnuclear area of the acrosome (Fig. 14b).

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◗ Damaged apical ridge (DAR): sperm are identified by the presence of particles in the area posterior to the apical ridge of the acrosome (damaged), which lends them an irregular shape (Fig. 14c). ◗ Missing apical ridge (MAR): sperm have lost the apical ridge, but part of the acrosome remains firmly attached to the head (Figs. 14d, 16, and 18). ◗ Loose acrosomal cap (LAC): characterised by the formation of vesicles in the anterior part of the acrosome (Figs. 14e, 16, and 17).

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EVALUATION OF SEMEN QUALITY

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Figure 14. Categorisation of acrosome integrity: normal apical ridge (NAR) (a), normal apical ridge with particles (NAR’) (b), damaged apical ridge (DAR) (c), missing apical ridge (MAR) (d), and loose acrosomal cap (LAC) (e).

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6

Semen processing and storage

Semen collection and processing

Preparation of semen doses and semen storage The semen should be gently added to the extender to prevent damage to the spermatozoa (Fig. 4).

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SEMEN PROCESSING AND STORAGE

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Figure 4. Semen and extender are gently mixed. Initial (a) and intermediate (b) steps.

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Semen processing and storage

Semen collection and processing

Extension rate To obtain a desired insemination dose (or bottle) of 3 × 109 spermatozoa/100 ml, the amount of extender required should be calculated as follows: 70

Total number of spermatozoa

Number of doses at required concentration

Determined sperm concentration (see Chapter 5) × Ejaculate volume

Total number of spermatozoa ÷ Desired extension rate

52 × 107 spermatozoa/ml × 125 ml = 65 × 109 total spermatozoa

65 × 109 spermatozoa ÷ 3 × 109 spermatozoa/dose = 21 doses

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SEMEN PROCESSING AND STORAGE

Total volume required

Extender volume

Number of doses ×Volume of semen dose

Total volume – Ejaculate volume

21 × 100 ml = 2,100 ml total volume

2,100 ml – 125 ml = 1,975 ml of extender required 71

Therefore: ◗ Add 125 ml semen to 1,975 ml of extender. ◗ Transfer extended semen into 21 bottles (100 ml) to obtain 3 × 109 spermatozoa/dose. ◗ Finally, the temperature of the semen dose is gradually decreased from 20–25 °C to 15–17 °C over 90 to 150 minutes for subsequent refrigerated storage or transport (Fig. 7).

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