Semen evaluation in domestic animals I

Transcription

1 Reproductive Biotechnologies Andrology I I Semen evaluation in domestic animals I Prof. Alberto Contri

2 Different aims 1. Diagnosis 2. Handling and preservation

3 Related to the male - Breeding soundness evaluation - Evaluation of the male for the function development related to the breed and age evaluation of the ability to mount and behavior semen evaluation

4 Development related to the breed and age Nutritional state and management Functional integrity of the limbs forelimbs hindlimbs

5 Development related to the breed and age Examination and palpation of the scrotum and its content normal morphology and shape normal consistence

6 Development related to the breed and age Scrotal circumference - bull Standards at puberty (28-30 cm) Highly heritable - selection

7 Testicular volume Related to the age and breed High correlation with testicular weigth High correlation with testicular parenchyma Daily production of spermatozoa (spz/die=17m die/g parenchima)

8 Scrotal circumference

9 Testicular parenchyma Volume = 4/3π x width/2 x height/2 x length/2 For each testis calculate the total testicular volume High related to the daily sperm output (DSO) DSO (x10 9 )=0.024 (TTV) 1.26

10 Ability to mount and normal behavior Libido - Instinct to copulation Calculation of the reaction time Heritable characteristic Initially related to hormone Training and reflex

11 Ability to mount and normal behavior Interaction with the female Sniff of the genital tract Flehmen reflex Erection Ejaculation

12 Ability to mount and normal behavior Ejaculation time Tom few seconds bull 3-5 seconds horse 1 minute or more (to 8-12 min) Boar several minutes Dog several minutes

13 Normal schedule for semen collection Training different for species months horse 12 months bull 8-12 months dog Until 2 years 1 collection/week Over 2 years 2 collections/week Intensive collections (16/month with rest)

14 Semen evaluation Gross inspection volume (different methods related to the volume) colour smell ph differ in different species texture - fluidity consistence Dog Bull Horse

15 Semen evaluation Microscopic evaluation concentration motility morphology

16 Concentration Number of spermatozoa per volume unit Considered the stronghold of the semen evaluation

17 Concentration breeding soundness certification diagnosis/prognosis repro disease handling of semen con preservation assessment of treatment effects

18 Ejacuate= sperm + seminal plasma Seminal plasma = epididymal fluid accessory glands fluid Concentration = spz in the volume unit (ml) x10 6 spz in 60 ml = 260x10 6 spz/ml x10 6 spz in 120 ml = 130x10 6 spz/ml

19 Irrational to talk about concentration to define spermatogenetic efficiency Parameters for the testicular function and spermatogenetic efficiency Daily sperm production (DSP) / gram of parenchyma Total number of sperm (Tsperm) Number of sperm/h Daily sperm output

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21 Concentration measurement large laboratory interindividual variability 23%-73% CV 53%-80% CV 21%-34% CV

22 Concentration measurement factors affecting diluents - disperse sperm - do not interfer with counting method - sperm immobilization - prevent agglutination

23 Concentration measurement diluents immobilization distilled water 10% saline solution 30 mm sodium fluoride 0.35% formalin (tested) Other specific solutions (nucleocounter)

24 Concentration measurement pitfalls dilution of the sample small sample volume large dilution

25 Concentration measurement proper sample preparation pipettes air positive displacement standardize the method

26 Concentration measurement pipetting method operating botton pressed to the 1 stop immersion of the tip slow aspiration of the solution clean the tip by adsorbent paper press the botton (blow out) and release the sample

27 Concentration measurement automated method autodiluters autodispensers

28 Concentration measurement cell counting chamber (Burker, Thoma ) Estimation of sperm in a known volume

29 Concentration measurement Gold standard Limits Related to the sample preparation Time-consuming technique

30 Concentration measurement Count at least 400 spz to reduce the error below 5% Count 2 replicates/chamber (diff <10%) Count all sperm in 1 mm 2

31 Camera di Burker n x h x dil x conv

32 Concentration measurement

33 Concentration measurement

34 Concentration measurement Automated technique: 1) NucleoCounter (Reagent-S100) 2) Computer assisted sperm analyzer (CASA) 3) Electronic counters (Coulter Counter) 4) Spettrophotometers 5) Flow cytometer

35 Concentration measurement Nucleocounter SP100

36 Concentration measurement Nucleocounter SP100

37 Concentration measurement CASA

38 Concentration measurement Coulter counter principle

39 Concentration measurement Photometer Optical absorbance Specific correlation between sperm density and ABS

40 Concentration measurement Flow cytometry Problems related to the volume of analysis required the use of fluospheres Problems related to debris

41 Normal concentration bull Horse Dog 900 x 10 6 /ml ( x 10 6 /ml) 220 x 10 6 /ml ( x 10 6 /ml) not indicative ( x 10 6 /ml)

42 Sperm motility Semen evaluation in domestic animals I Subjective mass motility (0-100%) motility score (from 0 to 5)

43 Sperm motility Objective Sperm quality analyzer (SQA) Computer-assisted sperm analyzer (CASA)

44 Sperm motility SQA V record the difference in cell density Based on optical absorbance of a sample trough a capillar Measurement of: Sperm concentration Sperm motility

45 Sperm motility CASA system

46 Sperm motility CASA, hardwares: Negative phase contrast microscope High frequency camera Pc for data processing

47 Sperm motility CASA system how it works Detection of each head Centroid calculation Detection of the precise head in following frames (Tracking) Calculation of the path Calculation of several parameters

48 Sperm motility CASA system what is important Precise detection of the head - setting Anti-collision function Frame rate

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50 Kinetic parameters Average path velocity (VAP) Straight line velocity (VSL) Curvilinear velocity (VCL) Lateral head displacement (ALH) Beat cross frequency (BCF) Straightness (STR) Linearity (LIN) Elongation (ELONG) Head surface (SIZE)

51 CASA system Sample loaded in a chamber Differences related to the chamber

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54 Data available for the mean population the single track

55 CASA single track evaluation

56 Detection of sperm subpopulations

57 Sperm subpopulations Cluster 1 (mean± sd) Electroejaculated semen Cluster 2 (mean± sd) Cluster 3 (mean± sd) Cluster 1 (mean± sd) Epididymal semen Cluster 2 (mean± sd) Cluster 3 (mean± sd) % population VAP (μm/s) 53.56±47.11 a ±69.04 b 71.09±76.11 c 10.51±6.54 d ±57.98 e 76.33±56.49 de VSL (μm/s) 14.58±13.82 a ±65.96 b 59.38±71.66 c 7.49±5.65 d 91.19±55.84 e 50.05±53.95 f VCL (μm/s) ±70.42 a ±81.97 b ±96.28 c 24.58±15.96 d ±88.34 e ±88.4 de ALH (μm) 7.20±3.29 a 6.66±2.79 b 3.75±2.89 c 1.25±0.87 d 6.76±2.7 b 6.94±3.48 b BCF (Hz) 49.06±14.91 a 43.73±12.77 b 28.09±24.21 c 28.03±25.32 d 44.9±14.04 e 38.83±22.6 f STR (%) 29.05±13.49 a 78.47±17.69 b 72.64±21.55 c 69.87±24.3 d 77.60±19.16 e 52.35±29.75 d LIN (%) 10.13±4.85 a 51.21±21 b 43.08±25.03 c 38.06±26.3 d 43.25±18.12 e 27.02±22.63 d AREA (μm 2 ) 61.46±23.14 a 61.24±18.03 a 79.87±23.07 b 77.31±29.68 d 63.68±19.4 e 86.98± d ELONG (%) 4.85±3.8 a 6.13±4.42 b 2.64±2 c 2.24±2.02 d 6.05±2.91 b 2.41±2.56 d WOB (%) 36.82±12.86 a 63.25±17.51 b 55.82±20.7 c 50.87±22.82 d 53.80±14.71 e 45.25±17.41 d DANCE (μm 2 /s) ± a ± b ±845.0 c 39.39±37.61 d ± b ± d MeDANCE (%) 0.92±0.93 a 0.16±0.1 b 0.11±0.09 c 0.06±0.07 d 0.19±0.12 e 0.63±0.96 f

58 Sperm morphology Evaluation of the shape of spemratozoa on stained samples (Diff-Quik, or others) spread on a slide air dried stained after strong fixation on unstained samples (Hankock method)

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60 Sperm morphology different types of classification a) Related to the sperm portion affected 1. abn. of the head 2. abn. of the midpiece 3. abn. of the tail

61 Sperm morphology b) Related to the different process involved a. primary abn. (spermatogenesis) b. secondary abn. (maturation) c. tertiary abn. (post-ejaculation)

62 Sperm morphology c) Adverse effects on male fertility a. major defects (have consequence on fertility) b. minor defects (have not clear consequence on fertility)

63 Sperm morphology Semen evaluation in domestic animals I

64 Sperm morphology d) Compensable and uncompensable a. compensable failure to reach the fertilization site b. uncompensable inability to fertilize the oocyte

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66 Immature forms