Live and Dead Spermatozoa in Cattle: Principle and Sperm Abnormalities

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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 1 — TITLE
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// ════════════════════════════════════════════════════════════════════════════
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    { num: "03", title: "Interpretation of Results", sub: "Live / dead identification & pitfalls" },
    { num: "04", title: "Hypoosmotic Swelling Test", sub: "Alternative viability method" },
    { num: "05", title: "Sperm Abnormalities in Cattle", sub: "Classification systems & individual defects" },
    { num: "06", title: "Compensable vs. Uncompensable", sub: "Fertility implications" },
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 3 — WHY ASSESS SPERM VIABILITY?
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 4 — EOSIN-NIGROSIN: HISTORY & PRINCIPLE
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 5 — REAGENTS & PROCEDURE
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    { n: "3", text: "Mix gently and incubate ~30 seconds at 37 °C" },
    { n: "4", text: "Make a thin smear and allow to air-dry completely" },
    { n: "5", text: "Examine under bright-field microscope at ×400–1000" },
    { n: "6", text: "Count minimum 200 spermatozoa per slide" },
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 6 — INTERPRETATION & PITFALLS
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0, y: 0, w: 10, h: 5.625,
    fill: { color: C.offwhite },
    line: { color: C.offwhite, width: 0 },
  });
  leftBar(s, C.green);
  headerBand(s, "Interpretation of Results & Pitfalls", C.dark);

  // Summary table header
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0.35, y: 0.85, w: 9.3, h: 0.42,
    fill: { color: C.mid },
    line: { color: C.mid, width: 0 },
  });
  ["Cell Status", "Membrane", "Eosin Penetration", "Microscopic Appearance"].forEach((h, i) => {
    const xArr = [0.4, 2.4, 4.55, 7.0];
    s.addText(h, {
      x: xArr[i], y: 0.88, w: 2.1, h: 0.35,
      fontSize: 10, bold: true, color: C.white,
      fontFace: "Calibri", margin: 0, valign: "middle",
    });
  });

  // Row: Live
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0.35, y: 1.27, w: 9.3, h: 0.62,
    fill: { color: "E8F8F0" },
    line: { color: C.gray, width: 0.5 },
  });
  const liveRow = ["LIVE", "Intact (semi-permeable)", "EXCLUDED", "White / unstained head"];
  const liveColors = [C.green, C.text, C.green, C.green];
  liveRow.forEach((cell, i) => {
    const xArr = [0.4, 2.4, 4.55, 7.0];
    s.addText(cell, {
      x: xArr[i], y: 1.3, w: 2.1, h: 0.55,
      fontSize: 10, bold: i === 0 || i === 2, color: liveColors[i],
      fontFace: "Calibri", margin: 0, valign: "middle",
    });
  });

  // Row: Dead
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0.35, y: 1.89, w: 9.3, h: 0.62,
    fill: { color: "FFF0F0" },
    line: { color: C.gray, width: 0.5 },
  });
  const deadRow = ["DEAD", "Damaged / porous", "ENTERS CELL", "Pink to dark red head"];
  const deadColors = [C.red, C.text, C.red, C.red];
  deadRow.forEach((cell, i) => {
    const xArr = [0.4, 2.4, 4.55, 7.0];
    s.addText(cell, {
      x: xArr[i], y: 1.92, w: 2.1, h: 0.55,
      fontSize: 10, bold: i === 0 || i === 2, color: deadColors[i],
      fontFace: "Calibri", margin: 0, valign: "middle",
    });
  });

  // Pitfalls & Special Notes
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0.35, y: 2.65, w: 9.3, h: 2.75,
    fill: { color: C.white },
    line: { color: C.gray, width: 1 },
  });
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0.35, y: 2.65, w: 9.3, h: 0.42,
    fill: { color: C.dark },
    line: { color: C.dark, width: 0 },
  });
  s.addText("SPECIAL NOTES & PITFALLS", {
    x: 0.35, y: 2.65, w: 9.3, h: 0.42,
    fontSize: 11, bold: true, color: C.white,
    align: "center", valign: "middle", fontFace: "Calibri", margin: 0,
  });

  const notes = [
    { icon: "⚠", color: C.cream, text: "Leaky neck membrane (stain confined to neck only, head unstained) → Count as LIVE — not true membrane disintegration" },
    { icon: "✓", color: "E8F8F0", text: "Validation check: % Dead (EN) + % Motile (wet mount) must total < 100%. If >100%, a methodological error has occurred" },
    { icon: "❄", color: C.light, text: "Use warm slides (37 °C) — cold shock can cause false membrane damage, artificially inflating dead cell counts" },
    { icon: "⏱", color: C.cream, text: "~30 sec incubation is optimal — over-incubation increases false positives; under-incubation reduces contrast" },
  ];
  notes.forEach((n, i) => {
    const y = 3.15 + i * 0.54;
    s.addShape(pres.shapes.RECTANGLE, {
      x: 0.45, y, w: 0.4, h: 0.42,
      fill: { color: n.color },
      line: { color: C.gray, width: 0 },
    });
    s.addText(n.icon, {
      x: 0.45, y, w: 0.4, h: 0.42,
      fontSize: 14, align: "center", valign: "middle",
      fontFace: "Calibri", margin: 0,
    });
    s.addText(n.text, {
      x: 0.95, y: y + 0.04, w: 8.5, h: 0.38,
      fontSize: 9.5, color: C.text,
      fontFace: "Calibri", margin: 0, valign: "middle",
    });
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 7 — HYPOOSMOTIC SWELLING TEST
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0, y: 0, w: 10, h: 5.625,
    fill: { color: C.offwhite },
    line: { color: C.offwhite, width: 0 },
  });
  leftBar(s, C.mid);
  headerBand(s, "Alternative: Hypoosmotic Swelling (HOS) Test");

  // Principle box
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0.35, y: 0.85, w: 9.3, h: 1.6,
    fill: { color: C.white },
    line: { color: C.gray, width: 1 },
  });
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0.35, y: 0.85, w: 9.3, h: 0.42,
    fill: { color: C.mid },
    line: { color: C.mid, width: 0 },
  });
  s.addText("PRINCIPLE", {
    x: 0.35, y: 0.85, w: 9.3, h: 0.42,
    fontSize: 12, bold: true, color: C.white,
    align: "center", valign: "middle", fontFace: "Calibri", margin: 0,
  });
  s.addText(
    "A live sperm with an intact functional membrane placed in a hypoosmotic solution (~150 mOsm) will " +
    "absorb water by osmosis → the tail coils or curls (positive HOS).\n" +
    "A dead sperm with a non-functional membrane does NOT swell → tail remains straight (negative HOS).",
    {
      x: 0.5, y: 1.35, w: 9.0, h: 1.0,
      fontSize: 10, color: C.text,
      fontFace: "Calibri", margin: 0, wrap: true,
    }
  );

  // Two outcomes
  const outcomes = [
    { label: "POSITIVE HOS", sub: "(Live cell)", desc: "Intact membrane → Water influx → Tail coils/curls", color: C.green },
    { label: "NEGATIVE HOS", sub: "(Dead cell)", desc: "Damaged membrane → No swelling → Tail remains straight", color: C.red },
  ];
  outcomes.forEach((o, i) => {
    const x = 0.35 + i * 4.85;
    s.addShape(pres.shapes.RECTANGLE, {
      x, y: 2.6, w: 4.55, h: 2.8,
      fill: { color: C.white },
      line: { color: o.color, width: 2 },
      shadow: { type: "outer", color: "000000", blur: 4, offset: 2, angle: 135, opacity: 0.1 },
    });
    s.addShape(pres.shapes.RECTANGLE, {
      x, y: 2.6, w: 4.55, h: 0.52,
      fill: { color: o.color },
      line: { color: o.color, width: 0 },
    });
    s.addText(`${o.label}  ${o.sub}`, {
      x, y: 2.62, w: 4.55, h: 0.48,
      fontSize: 11, bold: true, color: C.white,
      align: "center", valign: "middle", fontFace: "Calibri", margin: 0,
    });
    s.addText(o.desc, {
      x: x + 0.15, y: 3.2, w: 4.25, h: 0.6,
      fontSize: 10, color: C.text,
      fontFace: "Calibri", margin: 0,
    });

    // Tail drawing (simplified)
    const baseX = x + 1.5;
    s.addShape(pres.shapes.ELLIPSE, {
      x: baseX, y: 3.9, w: 0.5, h: 0.3,
      fill: { color: o.color },
      line: { color: o.color, width: 0 },
    });
    if (i === 0) {
      // Coiled tail
      s.addShape(pres.shapes.RECTANGLE, {
        x: baseX + 0.5, y: 4.0, w: 0.9, h: 0.06,
        fill: { color: o.color },
        line: { color: o.color, width: 0 },
      });
      s.addShape(pres.shapes.ELLIPSE, {
        x: baseX + 1.2, y: 3.75, w: 0.5, h: 0.5,
        fill: { color: "FFFFFF", transparency: 100 },
        line: { color: o.color, width: 2 },
      });
      s.addText("(coiled)", {
        x: baseX, y: 4.45, w: 1.5, h: 0.3,
        fontSize: 9, color: C.green, italic: true,
        align: "center", fontFace: "Calibri", margin: 0,
      });
    } else {
      // Straight tail
      s.addShape(pres.shapes.RECTANGLE, {
        x: baseX + 0.5, y: 4.0, w: 1.4, h: 0.06,
        fill: { color: o.color },
        line: { color: o.color, width: 0 },
      });
      s.addText("(straight)", {
        x: baseX, y: 4.45, w: 1.5, h: 0.3,
        fontSize: 9, color: C.red, italic: true,
        align: "center", fontFace: "Calibri", margin: 0,
      });
    }
  });

  // Note box
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0.35, y: 5.25, w: 9.3, h: 0.25,
    fill: { color: C.cream },
    line: { color: C.gray, width: 0.5 },
  });
  s.addText("Note: HOS test is less precise for cattle compared to eosin-nigrosin staining — EN remains the gold standard.", {
    x: 0.5, y: 5.26, w: 9.0, h: 0.22,
    fontSize: 8.5, color: C.subtext, italic: true,
    fontFace: "Calibri", margin: 0, valign: "middle",
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 8 — CLASSIFICATION OF SPERM ABNORMALITIES
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0, y: 0, w: 10, h: 5.625,
    fill: { color: C.offwhite },
    line: { color: C.offwhite, width: 0 },
  });
  leftBar(s, C.red);
  headerBand(s, "Classification of Bovine Sperm Abnormalities");

  const systems = [
    { title: "Primary / Secondary", author: "Blom, 1950", desc: "Based on SITE OF ORIGIN\nPrimary = Spermatogenesis (testis)\nSecondary = Post-testicular (epididymis, ejaculation)", color: C.dark },
    { title: "Major / Minor", author: "Blom, 1971", desc: "Based on SIGNIFICANCE to fertility\nMajor defects = seriously impair fertility\nMinor defects = minor or no effect", color: C.mid },
    { title: "Compensable / Uncompensable", author: "Saacke, 1990", desc: "Based on MODE OF ACTION\nCompensable = more sperm overcomes defect\nUncompensable = increasing sperm numbers CANNOT compensate", color: C.red },
    { title: "Anatomical Location", author: "Descriptive", desc: "Based on STRUCTURAL SITE\nHead defects | Midpiece defects\nTail defects | Cytoplasmic droplets", color: C.green },
  ];
  systems.forEach((sys, i) => {
    const col = i % 2;
    const row = Math.floor(i / 2);
    const x = 0.3 + col * 4.85;
    const y = 0.85 + row * 2.4;

    s.addShape(pres.shapes.RECTANGLE, {
      x, y, w: 4.6, h: 2.2,
      fill: { color: C.white },
      line: { color: C.gray, width: 1 },
      shadow: { type: "outer", color: "000000", blur: 4, offset: 2, angle: 135, opacity: 0.1 },
    });
    s.addShape(pres.shapes.RECTANGLE, {
      x, y, w: 4.6, h: 0.5,
      fill: { color: sys.color },
      line: { color: sys.color, width: 0 },
    });
    s.addText(`${sys.title}`, {
      x: x + 0.1, y: y + 0.02, w: 3.8, h: 0.46,
      fontSize: 11, bold: true, color: C.white,
      fontFace: "Calibri", margin: 0, valign: "middle",
    });
    s.addText(sys.author, {
      x: x + 3.7, y: y + 0.08, w: 0.8, h: 0.32,
      fontSize: 8, color: "FFFFFFBB", italic: true,
      align: "right", fontFace: "Calibri", margin: 0, valign: "middle",
    });
    s.addText(sys.desc, {
      x: x + 0.15, y: y + 0.58, w: 4.3, h: 1.55,
      fontSize: 9.5, color: C.text,
      fontFace: "Calibri", margin: 0, wrap: true,
    });
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 9 — PRIMARY ABNORMALITIES
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0, y: 0, w: 10, h: 5.625,
    fill: { color: C.offwhite },
    line: { color: C.offwhite, width: 0 },
  });
  leftBar(s, C.dark);
  headerBand(s, "Primary Abnormalities — Origin: Spermatogenesis (Testis)");

  s.addText("These are considered MAJOR defects with significant effect on fertility", {
    x: 0.35, y: 0.82, w: 9.3, h: 0.35,
    fontSize: 10, italic: true, color: C.subtext,
    fontFace: "Calibri", margin: 0,
  });

  const primaries = [
    { name: "Underdeveloped / Small Heads", detail: "Immature nucleus; poor chromatin condensation; indicates spermatogenic failure" },
    { name: "Double Forms", detail: "Bicephalic (2 heads) or biflagellate (2 tails) — result of failed cytokinesis" },
    { name: "Knobbed / Missing Acrosome", detail: "Cannot penetrate zona pellucida — one of the most fertility-damaging defects (uncompensable)" },
    { name: "Narrow / Pyriform Heads", detail: "Tapered or pear-shaped heads — associated with poor DNA integrity" },
    { name: "Crater / Diadem Defect", detail: "Nuclear vacuoles appearing as craters — linked to chromatin instability and early embryonic death" },
    { name: "Proximal Cytoplasmic Droplet", detail: "Retained cytoplasmic remnant near neck — indicates testicular immaturity or thermoregulatory failure" },
    { name: "Abnormal Midpiece", detail: "Coiled, bent, or thickened midpiece — impairs motility and energy production" },
    { name: "Accessory Tails", detail: "Extra axonemal structures; likely genetic; may impair fertility" },
    { name: "Strongly Coiled Tails", detail: "Tight coiling from deep testicular/epididymal origin — distinguished from distal coil" },
  ];

  primaries.forEach((p, i) => {
    const col = i % 3;
    const row = Math.floor(i / 3);
    const x = 0.3 + col * 3.2;
    const y = 1.22 + row * 1.45;

    s.addShape(pres.shapes.RECTANGLE, {
      x, y, w: 3.05, h: 1.32,
      fill: { color: C.white },
      line: { color: C.gray, width: 0.5 },
    });
    s.addShape(pres.shapes.RECTANGLE, {
      x, y, w: 0.18, h: 1.32,
      fill: { color: C.dark },
      line: { color: C.dark, width: 0 },
    });
    s.addText(p.name, {
      x: x + 0.26, y: y + 0.06, w: 2.72, h: 0.4,
      fontSize: 9.5, bold: true, color: C.dark,
      fontFace: "Calibri", margin: 0,
    });
    s.addText(p.detail, {
      x: x + 0.26, y: y + 0.46, w: 2.72, h: 0.82,
      fontSize: 8.5, color: C.subtext,
      fontFace: "Calibri", margin: 0, wrap: true,
    });
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 10 — SECONDARY ABNORMALITIES
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0, y: 0, w: 10, h: 5.625,
    fill: { color: C.offwhite },
    line: { color: C.offwhite, width: 0 },
  });
  leftBar(s, C.mid);
  headerBand(s, "Secondary Abnormalities — Origin: Epididymis / Post-Ejaculation");

  s.addText("Generally considered MINOR defects — arise during epididymal transit, handling, or processing", {
    x: 0.35, y: 0.82, w: 9.3, h: 0.35,
    fontSize: 10, italic: true, color: C.subtext,
    fontFace: "Calibri", margin: 0,
  });

  const secondaries = [
    { name: "Small Normal Heads", detail: "Slightly reduced but normally shaped — minor significance" },
    { name: "Giant / Short Broad Heads", detail: "Abnormal nuclear size from epididymal malfunction" },
    { name: "Detached / Loose Acrosomal Membranes", detail: "Acrosome partially lifted — not fully disrupted; less severe than primary acrosome defects" },
    { name: "Abaxial Implantation", detail: "Tail inserts off-center at head base; mostly normal variation; extreme cases swim in circles" },
    { name: "Distal Cytoplasmic Droplet", detail: "Droplet migrated to principal piece — minor significance; most bulls have some proportion" },
    { name: "Detached Heads", detail: "Head separates from tail during processing/cold shock — artefactual in many cases" },
    { name: "Simple Bent Tail", detail: "Single bend; often from cold shock during semen handling — artefactual" },
    { name: "Distal Coiled Tail", detail: "Coiling at terminal piece — hypoosmotic stress; less serious than proximal coil" },
  ];

  secondaries.forEach((p, i) => {
    const col = i % 4;
    const row = Math.floor(i / 4);
    const x = 0.25 + col * 2.4;
    const y = 1.22 + row * 2.1;

    s.addShape(pres.shapes.RECTANGLE, {
      x, y, w: 2.25, h: 1.9,
      fill: { color: C.white },
      line: { color: C.gray, width: 0.5 },
    });
    s.addShape(pres.shapes.RECTANGLE, {
      x, y, w: 2.25, h: 0.38,
      fill: { color: C.mid },
      line: { color: C.mid, width: 0 },
    });
    s.addText(p.name, {
      x: x + 0.08, y: y + 0.04, w: 2.1, h: 0.32,
      fontSize: 8.5, bold: true, color: C.white,
      fontFace: "Calibri", margin: 0, valign: "middle",
    });
    s.addText(p.detail, {
      x: x + 0.08, y: y + 0.44, w: 2.1, h: 1.38,
      fontSize: 8, color: C.subtext,
      fontFace: "Calibri", margin: 0, wrap: true,
    });
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 11 — COMPENSABLE vs UNCOMPENSABLE
// ════════════════════════════════════════════════════════════════════════════
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  const s = pres.addSlide();
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0, y: 0, w: 10, h: 5.625,
    fill: { color: C.offwhite },
    line: { color: C.offwhite, width: 0 },
  });
  leftBar(s, C.red);
  headerBand(s, "Compensable vs. Uncompensable Sperm Defects");

  // Compensable column
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0.3, y: 0.85, w: 4.4, h: 4.55,
    fill: { color: C.white },
    line: { color: C.green, width: 2 },
    shadow: { type: "outer", color: "000000", blur: 5, offset: 3, angle: 135, opacity: 0.1 },
  });
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0.3, y: 0.85, w: 4.4, h: 0.5,
    fill: { color: C.green },
    line: { color: C.green, width: 0 },
  });
  s.addText("COMPENSABLE DEFECTS", {
    x: 0.3, y: 0.87, w: 4.4, h: 0.46,
    fontSize: 12, bold: true, color: C.white,
    align: "center", valign: "middle", fontFace: "Calibri", margin: 0,
  });
  s.addText(
    "Definition:\nDefective sperm FAIL to fertilize but do NOT block competing normal sperm. " +
    "Increasing the number of inseminated sperm can overcome reduced fertility.\n\n" +
    "Examples:\n• Detached heads\n• Distal cytoplasmic droplets\n• Distal coiled tails\n• Abaxial implantation\n• Simple bent tails\n\n" +
    "Clinical relevance:\nMore significant in AI (fixed sperm dose) than in natural service where bull can inseminate repeatedly.",
    {
      x: 0.45, y: 1.45, w: 4.1, h: 3.85,
      fontSize: 10, color: C.text,
      fontFace: "Calibri", margin: 0, wrap: true,
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  );

  // Uncompensable column
  s.addShape(pres.shapes.RECTANGLE, {
    x: 5.3, y: 0.85, w: 4.4, h: 4.55,
    fill: { color: C.white },
    line: { color: C.red, width: 2 },
    shadow: { type: "outer", color: "000000", blur: 5, offset: 3, angle: 135, opacity: 0.1 },
  });
  s.addShape(pres.shapes.RECTANGLE, {
    x: 5.3, y: 0.85, w: 4.4, h: 0.5,
    fill: { color: C.red },
    line: { color: C.red, width: 0 },
  });
  s.addText("UNCOMPENSABLE DEFECTS", {
    x: 5.3, y: 0.87, w: 4.4, h: 0.46,
    fontSize: 12, bold: true, color: C.white,
    align: "center", valign: "middle", fontFace: "Calibri", margin: 0,
  });
  s.addText(
    "Definition:\nDefective sperm ACTIVELY INTERFERE with normal sperm function — occupying binding sites without fertilizing. " +
    "Increasing sperm numbers CANNOT improve fertility.\n\n" +
    "Examples:\n• Knobbed / missing acrosome\n• Nuclear vacuoles (diadem defect)\n• Certain abnormal head shapes\n• Decapitated sperm defect\n• Dag defect (coiled midpiece)\n\n" +
    "Clinical relevance:\nParticularly dangerous in natural mating. These bulls should be classified UNSATISFACTORY regardless of sperm numbers.",
    {
      x: 5.45, y: 1.45, w: 4.1, h: 3.85,
      fontSize: 10, color: C.text,
      fontFace: "Calibri", margin: 0, wrap: true,
    }
  );

  // Arrow in between
  s.addText("vs.", {
    x: 4.7, y: 2.8, w: 0.6, h: 0.6,
    fontSize: 18, bold: true, color: C.gray,
    align: "center", valign: "middle", fontFace: "Calibri", margin: 0,
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 12 — SPECIFIC NOTABLE DEFECTS IN CATTLE
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  s.addShape(pres.shapes.RECTANGLE, {
    x: 0, y: 0, w: 10, h: 5.625,
    fill: { color: C.offwhite },
    line: { color: C.offwhite, width: 0 },
  });
  leftBar(s, C.dark);
  headerBand(s, "Clinically Notable Sperm Defects in Cattle");

  const defects = [
    {
      name: "Dag Defect",
      sub: "Coiled midpiece",
      body: "Genetic defect seen in certain breeds (e.g., Guernsey). Severely impairs motility. Bull should be culled.",
      color: C.dark,
    },
    {
      name: "Decapitated Sperm",
      sub: "Head detaches in epididymis",
      body: "Breed-associated (Guernsey bulls). Heads and tails separate spontaneously. Causes severe subfertility.",
      color: C.red,
    },
    {
      name: "Knobbed Acrosome",
      sub: "Acrosome abnormality",
      body: "Most fertility-impairing primary defect. Sperm cannot penetrate zona pellucida. Uncompensable — no cure.",
      color: C.red,
    },
    {
      name: "Nuclear Vacuoles",
      sub: "Diadem / Crater defect",
      body: "Linked to chromatin instability and sperm DNA fragmentation. Associated with early embryonic death post-fertilization.",
      color: C.dark,
    },
    {
      name: "Proximal Droplet",
      sub: "Cytoplasmic retention",
      body: "Indicates testicular immaturity or recent fever/scrotal insulation. Resolves if underlying cause is treated — monitor for 60 days.",
      color: C.mid,
    },
    {
      name: "Abaxial Tail",
      sub: "Off-center implantation",
      body: "Generally normal variation. In extreme angles, affected sperm swim in tight circles — then classified as abnormal.",
      color: C.green,
    },
  ];

  defects.forEach((d, i) => {
    const col = i % 3;
    const row = Math.floor(i / 3);
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    const y = 0.85 + row * 2.4;

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}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 13 — BSE CLASSIFICATION TABLE
// ════════════════════════════════════════════════════════════════════════════
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    ["Morphologically Normal Sperm", "≥ 70% normal morphology", "Core criterion — failure = Unsatisfactory"],
    ["Live Spermatozoa (EN stain)", "≥ 70% viable (live, unstained)", "Confirms viability alongside morphology"],
    ["Scrotal Circumference", "Age-dependent minimum (e.g., ≥ 30 cm at 15 mo)", "Correlates with daily sperm output"],
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    { label: "UNSATISFACTORY\nPOTENTIAL BREEDER", color: C.red, crit: "One or more criteria failed" },
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 14 — SUMMARY / CONCLUSION
// ════════════════════════════════════════════════════════════════════════════
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  const points = [
    { icon: "01", text: "Eosin-nigrosin (Blom, 1950) is the gold-standard viability stain — principle = dye exclusion based on membrane integrity" },
    { icon: "02", text: "Live sperm exclude eosin (white head); dead sperm absorb eosin (pink/red head); nigrosin provides dark contrast background" },
    { icon: "03", text: "≥ 70% live, morphologically normal sperm is required for a Satisfactory Potential Breeder classification in BSE" },
    { icon: "04", text: "Primary abnormalities arise in the testis (spermatogenesis) and are generally major; secondary arise post-testiculary and are generally minor" },
    { icon: "05", text: "Uncompensable defects (e.g., knobbed acrosome, diadem defect) cannot be overcome by increasing sperm numbers — most clinically dangerous" },
    { icon: "06", text: "Specific cattle defects — Dag, decapitated sperm, knobbed acrosome, proximal droplet — have distinct etiologies and varying prognoses" },
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  s.addText("Reference: Barth & Oko (1989). Abnormal Morphology of Bovine Spermatozoa. Iowa State University Press.", {
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// ── WRITE FILE ───────────────────────────────────────────────────────────────
pres.writeFile({ fileName: "/home/daytona/workspace/sperm-cattle-ppt/Live_Dead_Spermatozoa_Cattle.pptx" })
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  .catch(err => { console.error("ERROR:", err); process.exit(1); });

const pptxgen = require("pptxgenjs");
const pres = new pptxgen();
pres.layout = "LAYOUT_16x9";
pres.title = "Live and Dead Spermatozoa in Cattle";

// ── PALETTE (all 6-digit hex, no alpha) ─────────────────────────────────────
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  light:   "E8F5F8",   // pale teal
  cream:   "F5EDD6",   // warm cream
  red:     "C94040",   // coral red  = dead
  green:   "2E7D52",   // forest green = live
  white:   "FFFFFF",
  offwhite:"F7F9F9",
  text:    "1A2E35",
  sub:     "3D6B77",
  gray:    "B0C4CC",
  lgray:   "E2EAEC",
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// ── HELPERS ──────────────────────────────────────────────────────────────────
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 1 — TITLE
// ════════════════════════════════════════════════════════════════════════════
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  // decorative circles on right panel
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  txt(s,"LIVE", 6.75, 0.72, 1.35, 0.5,
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  txt(s,"Veterinary Reproductive Biology", 0.4, 5.1, 5.7, 0.35,
    { fontSize:10, color:C.gray });
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 2 — AGENDA
// ════════════════════════════════════════════════════════════════════════════
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  const items = [
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    ["03","Reagents & Procedure","One-step method, step-by-step"],
    ["04","Interpretation & Pitfalls","Live vs. dead; special cases"],
    ["05","Sperm Abnormality Classification","4 classification systems"],
    ["06","Primary Abnormalities","Testicular origin; major defects"],
    ["07","Secondary Abnormalities","Post-testicular; minor defects"],
    ["08","Compensable vs. Uncompensable","Fertility implications & BSE outcomes"],
  ];
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}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 3 — WHY ASSESS SPERM VIABILITY
// ════════════════════════════════════════════════════════════════════════════
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  leftBar(s, C.green);
  hdr(s, "Why Assess Sperm Viability?");

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  txt(s,"≥ 70%", 3.6, 0.93, 2.8, 0.7,
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  txt(s,"Normal viable sperm\nfor Satisfactory Potential Breeder",
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    { fontSize:8.5, color:C.gray, align:"center" });

  // three reason cards
  const cards = [
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      title:"Breeding Soundness Exam",
      body:"Core evaluation of bull fertility before the breeding season. Includes motility, morphology, and viability assessment." },
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      title:"Artificial Insemination",
      body:"Viability must be confirmed in chilled and frozen-thawed semen. Dead sperm in AI doses reduce conception rates." },
    { x:6.99, color:C.red, icon:"DX",
      title:"Differential Diagnosis",
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 4 — EOSIN-NIGROSIN PRINCIPLE
// ════════════════════════════════════════════════════════════════════════════
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    {text:"Developed by ", options:{fontSize:9.5, color:C.text}},
    {text:"Blom (1950)", options:{fontSize:9.5, bold:true, color:C.dark}},
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    {text:"• Originally two-step; now one-step method\n", options:{fontSize:9.5, color:C.text}},
    {text:"• Gold standard ", options:{fontSize:9.5, bold:true, color:C.dark}},
    {text:"in veterinary & human reproductive labs\n", options:{fontSize:9.5, color:C.text}},
    {text:"• Validated against WHO semen standards (Bjorndahl et al., 2003)", options:{fontSize:9.5, color:C.text}},
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    {text:"  Semi-permeable → Eosin excluded → Head stays WHITE\n\n", options:{fontSize:9.5, color:C.text}},
    {text:"DAMAGED membrane (dead):\n", options:{fontSize:9.5, bold:true, color:C.red}},
    {text:"  Porous → Eosin penetrates → Head stains PINK/RED\n\n", options:{fontSize:9.5, color:C.text}},
    {text:"Nigrosin: ", options:{fontSize:9.5, bold:true, color:C.dark}},
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  // --- LIVE SPERM ---
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    { fontSize:10, bold:true, color:C.green, align:"center" });
  // head (white, green border)
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  // midpiece
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  // tail
  rect(s, 1.98, 3.98, 1.1, 0.06, C.green);
  // label
  txt(s,"Unstained head (white)", 0.55, 4.35, 3.0, 0.32,
    { fontSize:8.5, italic:true, color:C.green, align:"center" });

  // --- MEMBRANE COMPARISON ---
  txt(s,"MEMBRANE INTEGRITY", 3.75, 3.42, 2.5, 0.32,
    { fontSize:9, bold:true, color:C.sub, align:"center" });
  oval(s, 3.85, 3.8, 1.1, 1.1, C.light, C.green, 2.5);
  txt(s,"Intact\n✓", 3.85, 4.05, 1.1, 0.6,
    { fontSize:9, bold:true, color:C.green, align:"center" });
  oval(s, 5.1, 3.8, 1.1, 1.1, "FDEAEA", C.red, 2.5);
  txt(s,"Porous\n✗", 5.1, 4.05, 1.1, 0.6,
    { fontSize:9, bold:true, color:C.red, align:"center" });

  // --- DEAD SPERM ---
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    { fontSize:10, bold:true, color:C.red, align:"center" });
  oval(s, 6.3, 3.84, 0.7, 0.42, "FBBCBC", C.red, 2.5);
  rect(s, 6.99, 4.02, 0.55, 0.07, C.red);
  rect(s, 7.53, 3.98, 1.1, 0.06, C.red);
  txt(s,"Pink/red stained head", 6.2, 4.35, 3.0, 0.32,
    { fontSize:8.5, italic:true, color:C.red, align:"center" });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 5 — REAGENTS & PROCEDURE
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  hdr(s, "Reagents & Step-by-Step Procedure (One-Step Method)");

  // Reagents
  card(s, 0.3, 0.85, 3.55, 4.55, C.mid, "REAGENTS", 12);
  const reagents = [
    {name:"Eosin Y  (5%)", role:"Vital dye — stains dead cells pink/red", dot:C.red},
    {name:"Nigrosin  (10%)", role:"Background dye — dark contrast only", dot:C.dark},
    {name:"Distilled Water", role:"Solvent for stain preparation", dot:C.sub},
    {name:"Warm Slide (37 °C)", role:"Prevents cold-shock artefact", dot:C.mid},
  ];
  reagents.forEach((r,i) => {
    const y = 1.44 + i*1.0;
    rect(s, 0.46, y+0.14, 0.12, 0.62, r.dot);
    txt(s, r.name, 0.66, y+0.04, 3.05, 0.38,
      { fontSize:11, bold:true, color:C.text });
    txt(s, r.role, 0.66, y+0.44, 3.05, 0.34,
      { fontSize:9, italic:true, color:C.sub });
  });

  // Steps
  card(s, 4.1, 0.85, 5.55, 4.55, C.dark, "PROCEDURE", 12);
  const steps = [
    "Place a small drop of fresh semen on a warm slide (37 °C)",
    "Add an equal volume of eosin-nigrosin stain solution",
    "Mix gently and incubate approximately 30 seconds at 37 °C",
    "Make a thin smear and allow to air-dry completely",
    "Examine under bright-field microscope at ×400–1000",
    "Count a minimum of 200 spermatozoa per slide",
    "Record % unstained heads (live) and % pink heads (dead)",
  ];
  steps.forEach((step, i) => {
    const y = 1.42 + i * 0.56;
    oval(s, 4.26, y+0.04, 0.36, 0.36, C.mid);
    txt(s, String(i+1), 4.26, y+0.04, 0.36, 0.36,
      { fontSize:10, bold:true, color:C.white, align:"center", valign:"middle" });
    txt(s, step, 4.72, y+0.04, 4.75, 0.38,
      { fontSize:9.5, color:C.text, valign:"middle", wrap:true });
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 6 — INTERPRETATION & PITFALLS
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  bg(s, C.offwhite);
  leftBar(s, C.green);
  hdr(s, "Interpretation of Results & Pitfalls");

  // Result table header
  rect(s, 0.3, 0.84, 9.35, 0.44, C.mid);
  [["Cell Status",0.35,1.8],["Membrane Integrity",2.2,2.4],["Eosin Entry",4.65,1.6],["Microscopic Appearance",6.3,3.3]]
    .forEach(([h,x,w]) => txt(s, h, x, 0.87, w, 0.38,
      { fontSize:10, bold:true, color:C.white, valign:"middle" }));

  // Live row
  rect(s, 0.3, 1.28, 9.35, 0.6, "E8F8F0", C.lgray, 0.5);
  [["LIVE",C.green],["Intact (semi-permeable)",C.text],["EXCLUDED",C.green],["White / unstained head",C.green]]
    .forEach(([cell,color],i) => {
      const xs=[0.38,2.22,4.67,6.32]; const ws=[1.78,2.36,1.56,3.28];
      txt(s, cell, xs[i], 1.31, ws[i], 0.54,
        { fontSize:10, bold:i===0||i===2, color, valign:"middle" });
    });

  // Dead row
  rect(s, 0.3, 1.88, 9.35, 0.6, "FFF0F0", C.lgray, 0.5);
  [["DEAD",C.red],["Damaged / porous",C.text],["ENTERS CELL",C.red],["Pink to dark-red head",C.red]]
    .forEach(([cell,color],i) => {
      const xs=[0.38,2.22,4.67,6.32]; const ws=[1.78,2.36,1.56,3.28];
      txt(s, cell, xs[i], 1.91, ws[i], 0.54,
        { fontSize:10, bold:i===0||i===2, color, valign:"middle" });
    });

  // Pitfalls
  card(s, 0.3, 2.62, 9.35, 2.78, C.dark, "SPECIAL NOTES & PITFALLS", 11);
  const notes = [
    {icon:"⚠", col:C.cream, t:"Leaky neck membrane: stain confined only to neck, head unstained → Count as LIVE (not true cell death)"},
    {icon:"✓", col:C.light, t:"Validation: % Dead (EN) + % Motile (wet mount) must total < 100%. If it exceeds 100%, a method error has occurred"},
    {icon:"❄", col:C.light, t:"Use warm slides (37 °C) — cold shock damages membranes artificially, inflating the dead-cell percentage"},
    {icon:"⏱", col:C.cream, t:"Optimal incubation ~30 seconds. Over-incubation → false positives; under-incubation → poor contrast"},
  ];
  notes.forEach((n,i) => {
    const y = 3.18 + i*0.54;
    rect(s, 0.42, y, 0.38, 0.42, n.col, C.lgray, 0);
    txt(s, n.icon, 0.42, y, 0.38, 0.42,
      { fontSize:13, align:"center", valign:"middle" });
    txt(s, n.t, 0.9, y+0.04, 8.6, 0.38,
      { fontSize:9.5, color:C.text, valign:"middle", wrap:true });
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 7 — HYPOOSMOTIC SWELLING TEST
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  bg(s, C.offwhite);
  leftBar(s, C.mid);
  hdr(s, "Alternative Viability Method: Hypoosmotic Swelling (HOS) Test");

  card(s, 0.3, 0.84, 9.35, 1.62, C.mid, "PRINCIPLE", 12);
  txt(s,
    "A live sperm with an intact functional membrane placed in hypoosmotic solution (~150 mOsm) absorbs water by osmosis → " +
    "tail coils or curls (positive HOS).\nA dead sperm with a non-functional membrane does NOT swell → tail remains straight (negative HOS).",
    { x:0.45, y:1.38, w:9.1, h:1.0, fontSize:10, color:C.text, fontFace:"Calibri", margin:0, wrap:true });

  // Positive
  rect(s, 0.3, 2.62, 4.55, 2.78, C.white, C.green, 2);
  rect(s, 0.3, 2.62, 4.55, 0.5, C.green);
  txt(s,"POSITIVE HOS — Live Cell", 0.3, 2.64, 4.55, 0.46,
    { fontSize:11, bold:true, color:C.white, align:"center", valign:"middle" });
  txt(s,"Intact membrane → Water influx → Tail coils / curls\n\nResult: SPERM IS ALIVE",
    0.45, 3.2, 4.25, 1.0,
    { fontSize:10, color:C.text, wrap:true });
  // coiled tail drawing
  oval(s, 0.55, 4.35, 0.52, 0.32, C.green, C.green, 0);
  rect(s, 1.06, 4.47, 0.6, 0.06, C.green);
  oval(s, 1.6, 4.2, 0.48, 0.48, C.white, C.green, 2);
  txt(s,"(tail coiled = LIVE)", 0.5, 4.78, 3.5, 0.28,
    { fontSize:8.5, italic:true, color:C.green, align:"center" });

  // Negative
  rect(s, 5.1, 2.62, 4.55, 2.78, C.white, C.red, 2);
  rect(s, 5.1, 2.62, 4.55, 0.5, C.red);
  txt(s,"NEGATIVE HOS — Dead Cell", 5.1, 2.64, 4.55, 0.46,
    { fontSize:11, bold:true, color:C.white, align:"center", valign:"middle" });
  txt(s,"Damaged membrane → No swelling → Tail stays straight\n\nResult: SPERM IS DEAD",
    5.25, 3.2, 4.25, 1.0,
    { fontSize:10, color:C.text, wrap:true });
  // straight tail
  oval(s, 5.3, 4.35, 0.52, 0.32, C.red);
  rect(s, 5.81, 4.47, 1.35, 0.06, C.red);
  txt(s,"(tail straight = DEAD)", 5.25, 4.78, 3.5, 0.28,
    { fontSize:8.5, italic:true, color:C.red, align:"center" });

  // Note
  rect(s, 0.3, 5.38, 9.35, 0.22, C.cream, C.lgray, 1);
  txt(s,"Note: EN staining remains the gold standard for cattle — HOS is an alternative when staining is unavailable.",
    0.45, 5.39, 9.1, 0.2,
    { fontSize:8, italic:true, color:C.sub, valign:"middle" });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 8 — CLASSIFICATION SYSTEMS
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  bg(s, C.offwhite);
  leftBar(s, C.red);
  hdr(s, "Classification Systems for Bovine Sperm Abnormalities");

  const sys = [
    { title:"Primary / Secondary", auth:"Blom, 1950", col:C.dark,
      body:"Based on SITE OF ORIGIN\n• Primary = Spermatogenesis in testis\n• Secondary = Post-testicular (epididymis, ejaculation, handling)" },
    { title:"Major / Minor", auth:"Blom, 1971", col:C.mid,
      body:"Based on SIGNIFICANCE to fertility\n• Major defects — seriously impair or eliminate fertility\n• Minor defects — little or no effect on fertility" },
    { title:"Compensable vs Uncompensable", auth:"Saacke, 1990", col:C.red,
      body:"Based on MODE OF ACTION\n• Compensable — more sperm overcomes effect\n• Uncompensable — increasing numbers CANNOT compensate" },
    { title:"Anatomical Location", auth:"Descriptive", col:C.green,
      body:"Based on STRUCTURAL SITE\n• Head defects (shape, acrosome, nucleus)\n• Midpiece defects | Tail defects\n• Cytoplasmic droplets (proximal / distal)" },
  ];
  sys.forEach((sy,i) => {
    const col = i%2; const row = Math.floor(i/2);
    const x = 0.28 + col*4.88;
    const y = 0.86 + row*2.38;
    rect(s, x, y, 4.62, 2.2, C.white, C.lgray, 1);
    rect(s, x, y, 4.62, 0.5, sy.col);
    txt(s, sy.title, x+0.1, y+0.03, 3.7, 0.44,
      { fontSize:11, bold:true, color:C.white, valign:"middle" });
    txt(s, sy.auth, x+3.8, y+0.1, 0.75, 0.3,
      { fontSize:8, italic:true, color:C.lgray, align:"right" });
    txt(s, sy.body, x+0.15, y+0.58, 4.32, 1.55,
      { fontSize:9.5, color:C.text, wrap:true });
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 9 — PRIMARY ABNORMALITIES
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  bg(s, C.offwhite);
  leftBar(s, C.dark);
  hdr(s, "Primary Abnormalities — Origin: Spermatogenesis (Testis) — MAJOR Defects");

  const prims = [
    ["Underdeveloped / Small Heads","Immature nucleus; poor chromatin condensation; spermatogenic failure"],
    ["Double Forms","Bicephalic or biflagellate — failed cytokinesis during spermiogenesis"],
    ["Knobbed / Missing Acrosome","Cannot penetrate zona pellucida — most fertility-damaging; uncompensable"],
    ["Narrow / Pyriform Heads","Tapered or pear-shaped heads; poor DNA integrity; associated with infertility"],
    ["Crater / Diadem Defect","Nuclear vacuoles — chromatin instability → early embryonic death post-fertilization"],
    ["Proximal Cytoplasmic Droplet","Retained cytoplasm near neck — indicates testicular immaturity or heat stress"],
    ["Abnormal Midpiece","Coiled, bent, or thickened midpiece — impairs motility and ATP production"],
    ["Accessory Tails","Extra axonemal structures; usually genetic in origin; may impair fertility"],
    ["Strongly Coiled Tail","Tight proximal coiling — testicular origin; different from distal cold-shock coil"],
  ];
  prims.forEach((p,i) => {
    const col=i%3; const row=Math.floor(i/3);
    const x=0.28+col*3.22; const y=0.85+row*1.6;
    rect(s, x, y, 3.08, 1.52, C.white, C.lgray, 0.5);
    rect(s, x, y, 0.16, 1.52, C.dark);
    txt(s, p[0], x+0.24, y+0.06, 2.78, 0.42,
      { fontSize:9.5, bold:true, color:C.dark });
    txt(s, p[1], x+0.24, y+0.5, 2.78, 0.96,
      { fontSize:8.5, color:C.sub, wrap:true });
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 10 — SECONDARY ABNORMALITIES
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  bg(s, C.offwhite);
  leftBar(s, C.mid);
  hdr(s, "Secondary Abnormalities — Origin: Epididymis / Post-Ejaculation — MINOR Defects");

  const secs = [
    ["Small Normal Heads","Slightly reduced size, normal shape — minor significance"],
    ["Giant / Short Broad Heads","Abnormal nuclear size from epididymal malfunction"],
    ["Detached / Loose Acrosome","Partially lifted acrosomal membrane — less severe than primary acrosome defects"],
    ["Abaxial Implantation","Off-center tail attachment — mostly normal variation; extreme cases swim in circles"],
    ["Distal Cytoplasmic Droplet","Droplet migrated to principal piece — minor; common in young bulls"],
    ["Detached Heads","Separation of head from tail — often processing or cold-shock artefact"],
    ["Simple Bent Tail","Single tail bend — cold shock during handling; largely artefactual"],
    ["Distal Coiled Tail","Terminal piece coiling — hypoosmotic stress; less serious than proximal coil"],
  ];
  secs.forEach((p,i) => {
    const col=i%4; const row=Math.floor(i/4);
    const x=0.24+col*2.42; const y=0.85+row*2.38;
    rect(s, x, y, 2.28, 2.2, C.white, C.lgray, 0.5);
    rect(s, x, y, 2.28, 0.4, C.mid);
    txt(s, p[0], x+0.08, y+0.04, 2.12, 0.34,
      { fontSize:8.5, bold:true, color:C.white, valign:"middle", wrap:true });
    txt(s, p[1], x+0.1, y+0.48, 2.08, 1.65,
      { fontSize:8.5, color:C.sub, wrap:true });
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 11 — COMPENSABLE vs UNCOMPENSABLE
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  bg(s, C.offwhite);
  leftBar(s, C.red);
  hdr(s, "Compensable vs. Uncompensable Sperm Defects");

  // Compensable
  rect(s, 0.28, 0.84, 4.42, 4.6, C.white, C.green, 2);
  rect(s, 0.28, 0.84, 4.42, 0.5, C.green);
  txt(s,"COMPENSABLE", 0.28, 0.86, 4.42, 0.46,
    { fontSize:13, bold:true, color:C.white, align:"center", valign:"middle" });
  txt(s,[
    {text:"Definition:\n", options:{bold:true, fontSize:10, color:C.dark}},
    {text:"Defective sperm fail to fertilize but do NOT block competing normal sperm. ",options:{fontSize:10,color:C.text}},
    {text:"Increasing inseminated sperm numbers can overcome the fertility loss.\n\n",options:{fontSize:10,color:C.text}},
    {text:"Examples:\n", options:{bold:true, fontSize:10, color:C.green}},
    {text:"• Detached heads\n• Distal cytoplasmic droplets\n• Distal coiled tails\n• Abaxial implantation\n• Simple bent tails\n\n",options:{fontSize:10,color:C.text}},
    {text:"Clinical note:\n", options:{bold:true, fontSize:10, color:C.dark}},
    {text:"More critical in AI (fixed sperm dose) than in natural service.",options:{fontSize:10,color:C.sub}},
  ], { x:0.42, y:1.44, w:4.1, h:3.9, fontFace:"Calibri", margin:0, wrap:true });

  // Uncompensable
  rect(s, 5.3, 0.84, 4.42, 4.6, C.white, C.red, 2);
  rect(s, 5.3, 0.84, 4.42, 0.5, C.red);
  txt(s,"UNCOMPENSABLE", 5.3, 0.86, 4.42, 0.46,
    { fontSize:13, bold:true, color:C.white, align:"center", valign:"middle" });
  txt(s,[
    {text:"Definition:\n", options:{bold:true, fontSize:10, color:C.dark}},
    {text:"Defective sperm actively interfere with normal sperm function — occupying binding sites without fertilizing. ",options:{fontSize:10,color:C.text}},
    {text:"More sperm CANNOT compensate.\n\n",options:{fontSize:10,color:C.text}},
    {text:"Examples:\n", options:{bold:true, fontSize:10, color:C.red}},
    {text:"• Knobbed / missing acrosome\n• Nuclear vacuoles (diadem defect)\n• Certain abnormal head shapes\n• Decapitated sperm defect\n• Dag defect (coiled midpiece)\n\n",options:{fontSize:10,color:C.text}},
    {text:"Clinical note:\n", options:{bold:true, fontSize:10, color:C.dark}},
    {text:"Dangerous in natural mating — bull should be classified UNSATISFACTORY.",options:{fontSize:10,color:C.sub}},
  ], { x:5.44, y:1.44, w:4.1, h:3.9, fontFace:"Calibri", margin:0, wrap:true });

  txt(s,"vs.", 4.7, 2.85, 0.6, 0.55,
    { fontSize:20, bold:true, color:C.gray, align:"center", valign:"middle" });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 12 — NOTABLE SPECIFIC DEFECTS
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  bg(s, C.offwhite);
  leftBar(s, C.dark);
  hdr(s, "Clinically Notable Sperm Defects in Cattle");

  const defects = [
    {name:"Dag Defect",sub:"Coiled midpiece",body:"Genetic; seen in Guernsey & other breeds. Severely impairs motility. Affected bull should be culled.",col:C.dark},
    {name:"Decapitated Sperm",sub:"Head detaches in epididymis",body:"Breed-associated (Guernsey bulls). Heads and tails separate spontaneously during epididymal transit → severe subfertility.",col:C.red},
    {name:"Knobbed Acrosome",sub:"Primary acrosome defect",body:"Most fertility-impairing defect. Sperm cannot penetrate zona pellucida. Uncompensable — bull should fail BSE.",col:C.red},
    {name:"Nuclear Vacuoles",sub:"Diadem / Crater defect",body:"Chromatin instability and DNA fragmentation. Associated with fertilization failure and early embryonic death.",col:C.dark},
    {name:"Proximal Droplet",sub:"Cytoplasmic retention at neck",body:"Indicates immaturity or fever / scrotal insulation. Re-examine after 60 days (one full spermatogenic cycle) if cause identified.",col:C.mid},
    {name:"Abaxial Tail",sub:"Off-center insertion",body:"Usually a normal variation. Extreme cases cause circular swimming. Only abnormal when holding sperm at acute angle.",col:C.green},
  ];
  defects.forEach((d,i) => {
    const col=i%3; const row=Math.floor(i/3);
    const x=0.28+col*3.22; const y=0.84+row*2.4;
    rect(s, x, y, 3.06, 2.24, C.white, d.col, 1.5);
    rect(s, x, y, 3.06, 0.54, d.col);
    txt(s, d.name, x+0.1, y+0.04, 2.86, 0.3,
      { fontSize:11, bold:true, color:C.white });
    txt(s, d.sub, x+0.1, y+0.32, 2.86, 0.2,
      { fontSize:8.5, italic:true, color:C.lgray });
    txt(s, d.body, x+0.12, y+0.62, 2.82, 1.55,
      { fontSize:9, color:C.text, wrap:true });
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 13 — BSE CLASSIFICATION TABLE
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  bg(s, C.offwhite);
  leftBar(s, C.mid);
  hdr(s, "Bull Breeding Soundness Examination (BSE) — Classification Criteria");

  // Table header
  rect(s, 0.28, 0.84, 9.44, 0.46, C.dark);
  [["Parameter",0.34,2.6],["Minimum Requirement",2.96,3.3],["Classification Significance",6.3,3.38]]
    .forEach(([h,x,w]) => txt(s,h, x, 0.87, w, 0.4,
      { fontSize:10, bold:true, color:C.white, valign:"middle" }));

  const rows = [
    ["Gross Motility","≥ Fair  OR  ≥ 30% progressive motility","Must pass — below minimum = Unsatisfactory"],
    ["Morphologically Normal Sperm","≥ 70% normal morphology","Core criterion — most commonly failed"],
    ["Live Spermatozoa (EN stain)","≥ 70% viable (unstained by eosin)","Confirms vitality alongside morphology"],
    ["Scrotal Circumference","Age-dependent minimum (e.g. ≥ 30 cm at 15 mo)","Correlates with daily sperm output & capacity"],
  ];
  rows.forEach((row,ri) => {
    const y = 1.3 + ri*0.78;
    const bgc = ri%2===0 ? C.white : C.light;
    rect(s, 0.28, y, 9.44, 0.75, bgc, C.lgray, 0.5);
    [row[0],row[1],row[2]].forEach((cell,ci) => {
      const xs=[0.34,2.96,6.3]; const ws=[2.56,3.24,3.34];
      txt(s, cell, xs[ci], y+0.06, ws[ci], 0.62,
        { fontSize:9.5, bold:ci===0, color:ci===0?C.dark:C.text,
          valign:"middle", wrap:true });
    });
  });

  // Outcome boxes
  const outcomes = [
    {label:"SATISFACTORY\nPOTENTIAL BREEDER",col:C.green,note:"All minimum criteria met"},
    {label:"DEFERRED\n(Re-examine in 60 days)",col:C.mid,note:"Marginal results — one cycle recovery"},
    {label:"UNSATISFACTORY\nPOTENTIAL BREEDER",col:C.red,note:"One or more criteria failed"},
  ];
  outcomes.forEach((o,i) => {
    const x=0.28+i*3.18;
    rect(s, x, 4.5, 3.06, 0.94, o.col);
    txt(s, o.label, x+0.05, 4.53, 2.96, 0.52,
      { fontSize:10, bold:true, color:C.white, align:"center", valign:"middle" });
    txt(s, o.note, x+0.05, 5.04, 2.96, 0.35,
      { fontSize:8, italic:true, color:C.lgray, align:"center" });
  });
}

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 14 — SUMMARY
// ════════════════════════════════════════════════════════════════════════════
{
  const s = pres.addSlide();
  bg(s, C.dark);
  rect(s, 0, 4.85, 10, 0.775, C.mid);
  rect(s, 0, 4.82, 10, 0.06, C.cream);

  txt(s,"Key Takeaways", 0.45, 0.12, 9, 0.66,
    { fontSize:27, bold:true, color:C.white });
  txt(s,"Live & Dead Spermatozoa in Cattle", 0.45, 0.72, 9, 0.42,
    { fontSize:13, italic:true, color:C.cream });

  const pts = [
    "Eosin-nigrosin (Blom, 1950) is the gold-standard viability stain based on membrane dye-exclusion",
    "Live sperm exclude eosin (white head); dead sperm absorb it (pink/red head); nigrosin provides dark background contrast",
    "≥ 70% live, morphologically normal sperm is required for a Satisfactory Potential Breeder in BSE",
    "Primary abnormalities arise in the testis (major defects); secondary arise post-testicularly (generally minor)",
    "Uncompensable defects (knobbed acrosome, diadem) cannot be overcome by more sperm — most clinically dangerous",
    "Notable cattle defects: Dag, decapitated sperm, knobbed acrosome, proximal droplet — each with distinct etiology",
  ];
  pts.forEach((p,i) => {
    const col=i%2; const row=Math.floor(i/2);
    const x=0.32+col*4.88; const y=1.25+row*1.18;
    rect(s, x, y, 4.68, 1.08, "1E6678", "3BA8BB", 1);
    oval(s, x+0.1, y+0.32, 0.44, 0.44, C.mid);
    txt(s, String(i+1).padStart(2,"0"), x+0.1, y+0.32, 0.44, 0.44,
      { fontSize:9, bold:true, color:C.white, align:"center", valign:"middle" });
    txt(s, p, x+0.64, y+0.1, 3.96, 0.88,
      { fontSize:9, color:C.white, valign:"middle", wrap:true });
  });

  txt(s,"Reference: Barth & Oko (1989). Abnormal Morphology of Bovine Spermatozoa. Iowa State University Press.",
    0.3, 4.9, 9.4, 0.32,
    { fontSize:7.5, italic:true, color:C.lgray, align:"center", valign:"middle" });
}

// ── WRITE ───────────────────────────────────────────────────────────────────
pres.writeFile({ fileName: "/home/daytona/workspace/sperm-cattle-ppt/Live_Dead_Spermatozoa_Cattle_v2.pptx" })
  .then(() => console.log("SUCCESS"))
  .catch(err => { console.error("ERROR:", err); process.exit(1); });